TWI742008B - Immune modulation and treatment of solid tumors with antibodies that specifically bind cd38 - Google Patents

Abstract

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

Description

Use antibodies that specifically bind CD38 to immunomodulate and treat solid tumors [Cross-reference of related applications]

This application claims U.S. Application No. 15/191808 filed on June 24, 2016, International Application No. US16/39165 filed on June 24, 2016, and U.S. Provisional Application filed on May 4, 2016 No. 62/331,489, U.S. Provisional Application No. 62/263,307 filed on December 4, 2015, and U.S. Provisional Application No. 62/250,566 filed on November 4, 2015, and November 2, 2015 The rights and interests of the US Provisional Application No. 62/249,546, the entire contents of which are incorporated herein by reference.

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

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 provide a balanced network of positive and negative signals to maximize the immune response to infections and tumors, while limiting immunity to self (Wang et al., (Epub Mar. 7, 2011) J Exp Med 208(3): 577-92; Lepenies et al. (2008) Endocr Metab Immune Disord Drug Targets 8: 279-288).

The immune checkpoint therapy for the treatment of solid tumors (which targets the co-suppressive pathway in T cells to promote anti-tumor immune responses) is approved for anti-CTLA-4 and anti-PD-1 antibodies YERVOY ^® (ipilimumab), KEYTRUDA ^® (pembrolizumab) and OPDIVO ^® (nivolumab) have led to the advancement of clinical care for cancer patients. Although anti-PD-1/PD-L1 antibodies have been shown to promote clinical response in patients with a variety of solid tumors, the response rate is still quite low, which is 15% to 20% in pre-treated patients (Swaika et al. Human, (2015) Mol Immunol doi: 10.1016/j.molimm.2015.02.009).

Although natural killer cells (NK), dendritic cells (DC), and effector T cells can drive a strong anti-tumor response, tumor cells often induce an immunosuppressive microenvironment, which is conducive to the development of immunosuppressive immune cell populations. Such as bone marrow-derived suppressor cells (MDSC), regulatory T cells (Treg), or regulatory B cells (Breg), which lead to the failure of tumor immune tolerance and immunotherapy regimens in cancer patients and experimental tumor models.

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

The present invention provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need.

The present invention also provides a method for treating a disease mediated by regulatory T cells (Treg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to a patient in need of the disease.

The present invention also provides a method for treating a disease mediated by bone marrow-derived suppressor cells (MDSC), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need.

The present invention also provides a method for the treatment of a disease mediated by regulatory B cells (Breg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to a patient in need of the disease.

The present invention also provides a method for inhibiting the activity of regulatory T cells (Treg), which comprises contacting the Treg with an antibody that specifically binds to CD38.

The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSC), which comprises contacting the MDSC with an antibody that specifically binds to CD38.

The present invention also provides a method for inhibiting the activity of regulating B cells (Breg), which comprises contacting the Breg with an antibody that specifically binds to CD38.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering an antibody that specifically binds to CD38 to the patient.

The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of Treg cells in the patient by administering an antibody that specifically binds to CD38 to the patient.

The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of bone marrow-derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds to CD38.

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

The present invention also provides a method for treating a patient suffering from a viral infection, which comprises administering an antibody that specifically binds to CD38 to the patient in need.

Figure 1 shows that the median number of lymphocytes in the patient increases with time in response to ^{DARZALEX TM} (dalazumab) at a dose of 8 mg/kg (upper line) or 16 mg/kg (lower line). And after the end of treatment, the number of lymphocytes returned to the baseline. Research: SIRIUS. The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). SCR: base period; EOT: end of treatment; WK: weeks; POST-WK: designated weeks after treatment; post-PD FU: follow-up. The range highlighted in gray indicates that the interquartile range (IQR) of the data points for each visit of the respondent is 25 to 27%.

Figure 2 shows the percentage change (%) of the absolute counts ^{of CD3 +} T cells in the peripheral blood of patients treated ^{with DARZALEX TM} (dalazumab) for each individual patient (light gray line) from the baseline. Research: SIRIUS (MMY2002). The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). WK: week; POST-WK: specified number of weeks after treatment; POST-PD FU: follow-up after progress. The black line shows the% change in the median for all patients.

Figure 3 shows the percentage change (%) of the absolute count ^{of CD4 +} T cells in the peripheral blood of patients treated ^{with DARZALEX TM} (dalazumab) for each individual patient (light gray line) from the baseline. Research: SIRIUS. The X-axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C1D1: the first day of the first cycle; C1D4: the fourth day of the first cycle, etc.). WK: Week; POST-TMT: After treatment. The black line shows the% change in the median for all patients.

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

Figure 5 ^{shows that the number of CD45 +} CD3 ⁺ cells in bone marrow aspirates (measured as a percentage of lymphocytes) increased over time during treatment with ^DARZALEX™ (dalazumab) at a dose of 8 mg/kg or 16 mg/kg. The chart includes responders and non-responders as indicated. Research: SIRIUS. The X axis indicates the time, which is expressed in terms of the treatment cycle and the number of days of administration in each treatment cycle (C2D22: 22nd day of the second cycle; etc.). SCR: base period; Post-PD FU1: follow-up after progress. The range highlighted with gray shading indicates a quarter of the data points of each visit for non-responders who were dosed at 8 mg/kg, responders who were dosed at 16 mg/kg, or non-responders who were dosed at 16 mg/kg. The digit spacing (IQR) is 25 to 27%, respectively. NR: non-responders; R: responders.

Figure 6 shows that the number of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells in bone marrow aspirate (measured as a percentage of lymphocytes) increased over time during treatment with ^{DARZALEX TM} (dalazumab) at a dose of 8 mg/kg or 16 mg/kg . The chart includes responders and non-responders as indicated. Research: SIRIUS. The X axis indicates the time, which is represented by the treatment cycle and the number of days of administration in each treatment cycle (C2D22: 22nd day of the second cycle, etc.). SCR: base period; Post-PD FU1: follow-up after progress. The range highlighted with gray shading indicates a quarter of the data points of each visit for non-responders who were dosed at 8 mg/kg, responders who were dosed at 16 mg/kg, or non-responders who were dosed at 16 mg/kg. The digit spacing (IQR) is 25 to 27%, respectively. NR: non-responders; R: responders.

Figure 7A shows that during DARZALEX ^{TM (darumab) treatment, the ratio of CD8 +} /Treg and CD8 ⁺ /CD4 ⁺ cells in the peripheral blood represented by the median value of all treated patients increased. Time point: C1D1: the first day of the first cycle; C3D1: the first day of the third cycle; C4D1: the first day of the fourth cycle. Research: SIRIUS. SRC: Base period.

Fig. 7B shows that ^{the ratio of CD8 +} /Treg cells in the bone marrow aspirate expressed as the median value of all treated patients during ^{DARZALEX TM} (dalazumab) treatment increased over time. Time point: C1D1: the first day of the first cycle; C3D1: the first day of the third cycle; C4D1: the first day of the fourth cycle. Research: SIRIUS.

Figure 8A shows that when compared to non-responders, the CD8 ⁺ T cell lineage of responders increases, as measured using the change in abundance (CIA)% of specific lineage cells. Study: A subset of GEN501 17 patients.

Figure 8B shows the fold change ^{of CD8 +} T cell homologousness in individual patients before and after ^{DARZALEX TM (dalazumab) treatment.} Respondents are indicated by asterisks. Homogenous lineage is measured as a multiple of the change in abundance (CIA) of a specific inline cell line. Study: A subset of GEN501 17 patients.

Figure 8C shows that when compared to non-responders (group B), responders (group A) have a greater total amplification in the TCR reservoir, which is measured using CIA (change in abundance). P=0.037. Study: A subset of GEN501 17 patients.

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

Figure 8E shows the maximum CIA of single cell clones in responders (group A) and non-responders (group B). Study: A subset of GEN501 17 patients.

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

ve cell)的百分比(%)。研究：GEN501 17病患子集。**p=1.82×10^-4。 Figure 9A shows that in the baseline period, or after 2 weeks, 4 weeks, or 8 weeks of treatment, or after recurrence, non-responders (NR, black squares) and patients with at least the least response ^{to DARZALEX TM (dalazumab) (} MR, white square) CD8 ⁺ initial cells in the peripheral blood (na

ve cell) percentage (%). Study: A subset of GEN501 17 patients. **p=1.82×10 ^-4 .

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

Figure 9C ^{shows the percentage increase of HLA class I restricted CD8 +} T cells in the peripheral blood during the baseline period, or after 1, 4, or 8 weeks of treatment, or after recurrence. Study: A subset of GEN501 17 patients.

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

Figure 10A shows the distribution diagram of FACS analysis, which shows that in the base stage, ^{the frequency of Treg (CD3 +} CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ) in patients with multiple myeloma (upper distribution diagram, P4 cell population) and CD38 ⁺ Treg Frequency within the Treg population (distribution below, P5 cell population). Study: A subset of GEN501 17 patients.

Figure 10B shows the distribution diagram of FACS analysis, which shows the frequency ^{of Treg (CD3 +} CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ) in patients with multiple myeloma after ^{DARZALEX TM} (dalazumab) treatment (upper distribution diagram, P4 cell population) and ^{the frequency of CD38 +} Treg in the Treg population (distribution diagram below, P5 cell population). DARZALEX ^(TM ) treatment ^{depletes CD38 +} Treg. Study: A subset of GEN501 17 patients.

Figure 10C shows that in the base period, or at 1 week, 4 weeks, 8 weeks, after recurrence, or at the end of treatment (EOT) for 6 months, CD38 was ^{high in patients treated with DARZALEX TM (dalazumab).} CD4 ⁺ CD25 ⁺ CD127 ^dim Treg frequency. The frequency of CD38 ^high Treg decreased with DARZALEX ^TM (dalazumab) treatment, and returned to the baseline at EOT. Y axis:% of CD3 ⁺ T cell CD38 ^high CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim Treg. Study: A subset of GEN501 17 patients.

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

Figure 10E shows that when compared to CD38 ^- Treg or negative control, the proliferation of effector cells is more effectively inhibited in the presence of ^{CD38 + Treg.} Error bars represent standard errors. Asterisks indicate significant changes. The samples were obtained from multiple healthy donors. Cell proliferation was assessed by dilution of carboxyfluorescein succinimidyl ester (CFSE).

Figure 11 shows that bone marrow-derived suppressor cells (MDSC) are present in patients with multiple myeloma (top chart, boxed cells), and about half of the cells express CD38 (middle chart, boxed cells). The CD38 high MDSC population was depleted ^{in patients treated with a DARZALEX TM} (dalazumab) infusion (bottom chart, boxed cells). Study: A subset of GEN501 17 patients.

Figure 12 shows that when compared to the base period, after undergoing DARZALEX ^TM (Dara mAb) for 1 week, 4 weeks or 8 weeks, the patients in the high CD38 ^{MDSC (CD11b + HLADR - CD14 -} CD33 + CD15 + ) Decreased, and returned to near the base period after the end of treatment (EOT). Relapsed patients still showed a reduction in CD38 high MDSC. Black squares: non-responders; white squares: patients who have at least the least response ^{to DARZALEX TM (dalazumab) treatment.} The vertical line indicates the median value in each group. Patients 2, 4, 15, 16, and 17 exhibited a high initial CD38 high MDSC population. Study: A subset of GEN501 17 patients.

Figure 13 shows that patients with the highest CD38 high MDSC (patients 2, 4, 15, 16, and 17) have the highest progression-free survival (PFS). These patients have a partial response (PR) or minimal response (MR) ^{to DARZALEX TM (darumab) treatment.} SD: stable disease; PD: disease progression. The X axis shows the PFS of each individual patient.

Figure 14 shows that MDSC is sensitive to ADCC induced by ^{DARZALEX(TM).} Daudi cells are used as a positive control for target cells in the assay. Measure% cell lysis.

Figure 15A ^{shows that CD38 +} Breg was depleted in patients treated ^{with DARZALEX TM} (dalazumab) at the first week, the fourth week, and the eighth week of treatment.

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

Figure 16A shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated ^{with DARZALEX TM} (dalazumab) with VGPR at the baseline and at designated times during treatment Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4th, 8th, or 10th week of treatment.

Figure 16B shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated ^{with DARZALEX TM} (dalazumab) with CR at the baseline and at the designated time during treatment Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4th, 8th, or 10th week of treatment.

Figure 16C shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated ^{with DARZALEX TM} (dalazumab) with PD at the baseline and at the designated time during the treatment period Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Ns: Not significant. Pre 4,8 = 4th or 8th week of treatment.

Figure 16D shows the production of CMV, EBV, and influenza virus-specific (CEF) IFN-γ in the PBMC of patients treated ^{with DARZALEX TM} (dalazumab) at the base period and at the designated time during the treatment period. Antiviral response measured. OD: Optical density. White bar: negative control; black bar: CEF added; dashed bar: only allogeneic PBMC. Ns: Not significant. Pre 4,8 = 4th or 8th week of treatment.

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

Figure 16F shows the percentage (%) of proliferating virus-reactive T cells in the PBMC of patients treated ^{with DARZALEX™} (dalazumab) with CR at the base period and at the designated time during the treatment. White bar: negative control; black bar: CEF added. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4th, 8th, or 10th week of treatment.

Figure 17A shows a distribution diagram of FACS analysis, which shows the expression level of CD38 on natural killer cells (NK), monocytes, B cells, and T cells from healthy donors.

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

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

Figure 18 shows that PD-L1 protein is down-regulated in the PBMC samples of responders (R) and up-regulated in non-responders (NR) over time. SD: Stable disease. C1D1: the first day of the first cycle; C3D1: the first day of the third cycle. The Y axis shows the value of log2 protein concentration.

In the scope of this specification and the accompanying patent application, unless the content clearly indicates otherwise, the singular forms of "一 (a/an)" and "the (the)" include plural references. Therefore, for example, the reference to "a cell" includes a combination of two or more cells, and the like.

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

SEQ ID NO: 1

As used herein, "antibody" refers to and includes immunoglobulin molecules in a broad sense, including monoclonal antibodies (including murine, human, humanized, and chimeric monoclonal antibodies). Antibodies), antibody fragments, bispecific or multispecific antibodies, dimers, tetramers, or multimer antibodies, single chain antibodies, domain antibodies, and any other antigen binding sites that contain the desired specificity ( The modified configuration of the immunoglobulin of the antigen binding site).

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

"Antibody fragment" refers to a part of an immunoglobulin molecule that retains heavy and/or light chain antigen binding sites, such as heavy chain complementarity determining regions (HCDR) 1, 2, and 3, and light chain complementation Determining regions (LCDR) 1, 2, and 3, heavy chain variable region (VH), or light chain variable region (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, are contained in the hinge region connected by a disulfide bridge Two Fab fragments; Fd fragment, composed of VH and CH1 domains; Fv fragment, composed of VL and VH domains of one arm of an antibody; domain antibody (dAb) fragments (Ward et al., Nature 341:544-6, 1989), which is composed of the VH domain. The VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs, where the VH/VL domains are paired intramolecularly, or the VH and VL domains are separated by single chain antibodies In the case of constructs, intermolecular pairing will be performed to form a monovalent antigen binding site, such as a single chain Fv (scFv) or a diabody; for example, it is described in PCT International Publication No. WO1998/44001, No. WO1988 /01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using well-known techniques known to those with ordinary knowledge in the art, and these fragments are screened to have the same utility as full-length antibodies.

"Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to CD38 is substantially free of specific binding Antibodies to antigens other than human CD38). However, isolated antibodies that specifically bind to CD38 may cross-react with other antigens, such as heterologous human CD38, such as cynomolgus ( Macaca fascicularis , cynomolgus) CD38. In the case of bispecific antibodies, the bispecific antibody specifically binds to two antigens of interest, and is substantially free of antibodies that specifically bind to antigens that are not two antigens of interest. In addition, the isolated antibody may be substantially free of other cellular materials and/or chemicals. "Isolated antibody" covers antibodies that have been isolated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure antibody.

"Specific binding (specific binding)" or "bind" means that an antibody binds to an antigen or an epitope within the antigen with greater affinity than other antigens. Generally, the antibody is about 1×10 ^-8 M or less (e.g., about 1×10 ^-9 M or less, about 1×10 ^-10 M or less, about 1×10 ^-11 M or less, or The equilibrium dissociation constant (K _D ) of about 1×10 ^-12 M or less), which binds to an antigen or an epitope within an antigen, usually by the K of its binding to a non-specific antigen (eg, BSA, casein) _{K D} combination with _D at least one hundred times smaller. The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity with other related antigens, for example, for the same antigen (homolog) from another species (such as human or monkey), the monkey For example, crab-eating macaques ( Macaca fascicularis , cynomolgus, cyno), chimpanzees ( Pan troglodytes , chimpanzee, chimp), or marmosets ( Callithrix jacchus , common marmoset, marmoset). Although monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.

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

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

"Framwork" or "framwork sequence" refers to the remaining sequences in the variable region that are defined as the antigen binding site. Because the antigen binding site can be defined in various terms as described above, the exact amino acid sequence of the framework depends on how the antigen binding site is defined.

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

"Human antibody" refers to an antibody with heavy and light chain variable regions, in which both the framework and the antigen binding site are derived from human sequences. If the antibody contains a constant region, the constant region is also derived from human sequences.

A human antibody contains a heavy or light chain variable region "derived from" a human sequence, wherein the variable region of the antibody is obtained from the use of human germline immunoglobulin or rearranged immunoglobulin genes system. These systems include human immunoglobulin gene libraries displayed on phage, and gene transfer of non-human animals (such as mice with human immunoglobulin loci). "Human antibodies" may contain amino acid differences when compared with human germline immunoglobulin or rearranged immunoglobulin genes, due to, for example, naturally occurring somatic mutations or deliberate introduction in the framework or antigen binding site Replace, or both. Generally, "human antibodies" have at least about 80%, 81%, 82%, 83%, and the amino acid sequence encoded by the human germline immunoglobulin or rearranged immunoglobulin gene in the amino acid sequence. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % Identity. In some cases, "human antibodies" may contain consensus framework sequences derived from human framework sequence analysis, such as those described in Knappik et al., (2000) J Mol Biol 296:57-86, or incorporated into phage display Synthetic HCDR3 in the human immunoglobulin gene library, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO2009/085462.

Human antibodies derived from human immunoglobulin sequences can be produced using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or can be mutagenized in vitro to improve antibody properties, thereby obtaining in vivo Human antibodies are not naturally occurring antibodies in the repertoire of human antibodies.

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

"Recombinant antibody" includes all antibodies that are prepared, expressed, created, or isolated by recombinant means. Such as antibodies that are isolated from animals (such as mice or rats) or fusion tumors (described further below) prepared from gene transfer or chromosomal transfer of human immunoglobulin genes; self-transformation (transform) Antibodies isolated from host cells expressing antibodies; antibodies isolated from recombinant or combinatorial antibody libraries; and prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences Isolated antibodies; or antibodies such as bispecific antibodies produced in vitro using Fab arm exchange.

"Monoclonal antibody" refers to a preparation of antibody molecules composed of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope, or in the case of a bispecific monoclonal antibody, it has dual binding specificities for two different epitopes. A "monoclonal antibody" therefore refers to a group of antibodies with a single amino acid composition in each heavy chain and each light chain, except for possible well-known changes, such as the removal of the C-terminal lysine from the heavy chain of the antibody. Within the antibody population, monoclonal antibodies can have heterogeneous glycosylation. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. The bispecific antibody system is included in the term monoclonal antibody.

"Epitope" means the part of an antigen that specifically binds to an antibody. Epitopes are often composed of chemically active (such as polar, non-polar, or hydrophobic) surface groupings of molecular parts (such as amino acids or polysaccharide side chains), and can have specific three-dimensional structural characteristics and specific charges characteristic. Epitopes can be composed of contiguous and/or noncontiguous amino acids that form structural space units. Against non Adjacent to epitopes, amino acids from different parts of the linear sequence of the antigen will close together in a 3-dimensional space through the folding of protein molecules.

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

"In combination with" means that two or more therapeutic agents are administered to a subject together as a mixture, simultaneously administered in a single dose or sequentially administered in a single dose in any order. In general, each agent will be administered in a dose and/or according to a schedule determined for that agent.

"Treat (treat or treatment)" refers to therapeutic treatment, in which the purpose is to slow down (relieve) undesirable physiological changes or diseases, such as the development or spread of tumors or tumor cells, or to provide benefits or benefits in the course of treatment. Desired clinical results. Beneficial or desired clinical results include alleviation of symptoms, reduction of disease severity, stabilization of disease state (i.e., no deterioration), delay or slowdown of disease progression, no metastasis, improvement or alleviation of disease state, and Mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging the survival period compared to the expected survival period of an untreated subject. Those in need of treatment include those who have suffered undesired physiological changes or diseases, and those who are susceptible to such physiological changes or diseases.

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

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

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

"Effector T cell", or "Teffs_", or "Teff" refers to T lymphocytes that perform immune response functions, such as killing tumor cells and/or activating anti-tumor immune responses, which can cause tumors Cells are cleared from the body. Teff can be CD3 ⁺ and CD4 ⁺ or CD8 ⁺ . Teff can secrete, contain, or exhibit markers such as IFN-γ, granzyme B, and ICOS. It should be understood that Teffs may not be completely limited to these phenotypes.

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

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

"Myeloid-derived suppressor cells", or "MDSCs", or "MDSC" refer to the hematopoietic lineage and express the macrophage/monocyte marker CD11b and the particle marker Gr-1/Ly-6G The specialized cell population. The phenotype of MDSC can be, for example, CD11b ⁺ HLA-DR ^- CD14 ^- CD33 ⁺ CD15 ⁺ . MDSC shows low or undetectable performance of mature antigen-presenting cell markers MHC class II and F480. MDSC is an immature cell of the bone marrow lineage, and can be further differentiated into several cell types, including macrophages, neutrophils, dendritic cells, monocytes, or granulocytes. MDSC can be naturally found in normal adult bone marrow of humans and animals or in normal hematopoietic sites (such as the spleen).

"Inhibit function of MDSCs" or "inhibit MDSC function" refers to reducing the level of MDSC function in vitro or in an animal or human subject. Known conventional technology determination. The functional level of MDSC can be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "The function of inhibiting MDSC" includes reducing the number of MDSCs, for example by killing MDSCs through antibody effector functions (such as ADCC). MDSC can inhibit T cell response by various mechanisms (such as the production of active oxide peroxynitrite, the increase in sperminase metabolism due to high levels of sperminase, and the increase in nitrous oxide synthase) Such as hyperplasia, clone expansion, or cytokine production. MDSC can respond to IFN-γ and several cytokines such as IL-4 and IL-13. IFN-γ can activate MDSC, which induces the activity of nitric oxide synthase 2 (NOS2). Alternatively, Th2 cytokines such as interleukin-4 (IL-4) and IL-13 can activate MDSC, which can induce the activity of arginase-1 (ARG1). Metabolism of L-arginine by NOS2 or ARG1 can inhibit the proliferation of T cells, and the activities of the two enzymes together can lead to apoptosis of T cells through the production of active nitrogen oxides.

"Treg related disease (Treg related disease)" refers to a disease or disorder related to T regulatory cells (Treg). Treg-related diseases can be caused by Treg functions (e.g., suppression of anti-tumor response or suppression of effector T cell proliferation). Treg-mediated diseases can be cancer. "Treg-related disease" and "Treg mediated disease" are used interchangeably in this article.

"Enhance response of effector T cells" or "enhance T cell responses" refer to the enhancement or stimulation of effector T cells in vitro or in animal or human subjects to have sustained Or amplify biological functions, or renew or reactivate depleted or inactive T cells. Exemplary T cell response line proliferation, gamma interferon ^{secretion from CD 8 +} T cells, antigen reactivity, or pure line expansion. The way to measure this enhancement is known to those of ordinary knowledge in the art.

"MDSC related disease" refers to a disease or disorder related to bone marrow-derived suppressor cells (MDSC). MDSC-related diseases can be caused by MDSC functions (for example, inhibition of anti-tumor response or effector T cell proliferation). MDSC-mediated diseases can be cancer. "MDSC-related disease" and "Treg mediated disease" are used interchangeably in this article.

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

"Breg related disease" refers to a disease or disorder related to regulatory B cells. Breg-related diseases can be caused by, for example, Breg-mediated anti-tumor response suppression or effector T cell proliferation. Breg-mediated diseases can be cancer. "Breg related disease" and "Breg mediated disease" are used interchangeably in this article.

"Patient" includes any human or nonhuman animal. "Non-human animals" include all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and so on. "Patient" and "subject" are used interchangeably in this article.

The present invention provides a method for treating patients with solid tumors with antibodies that specifically bind CD38, regardless of whether the tumor cells express CD38. The present invention further provides methods for treating patients suffering from diseases mediated by regulatory T cells (Treg), bone marrow-derived suppressor cells (MDSC), or regulatory B cells (Breg). The present invention further provides methods for regulating the activity of Treg, MDSC, or Breg to treat solid tumors that are CD38 positive and/or are associated with high levels of these immunosuppressive cells.

The present invention is based at least in part on the discovery that the anti-CD38 antibody DARZALEX ^TM (daratumumab) has immunomodulatory activity in patients, which reduces the number of immunosuppressive Treg, MDSC, and Breg, and increases CD8 ⁺ The number of T cells and ^{the ratio of CD8 +} to Treg, promote the ^{formation of CD8 +} central memory cells, and increase the homologousness of T cells.

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

Without wishing to be bound by any particular theory, based on the immunomodulatory effects observed ^{with DARZALEX TM (darumab) described herein, DARZALEX TM} (darumab) and other anti-CD38 antibodies may be involved in the treatment of solid tumors. Effective. Due to the general activation of the immune response observed in patients treated with DARZALEX ^TM (darumab), patients with CD38-negative solid tumors may also respond to anti-CD38 antibody therapy.

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

The present invention also provides a method for treating a patient suffering from a disease mediated by regulatory T cells (Treg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient in need thereof for a period of time sufficient for treatment The time of the disease of the Treg vector.

The present invention also provides a method for treating a patient suffering from a disease mediated by bone marrow-derived suppressor cells (MDSC), which comprises administering to the patient in need a therapeutically effective amount of an antibody that specifically binds CD38 for a period of time sufficient The time to treat the disease of the MDSC vector.

The present invention also provides a method for treating a patient suffering from a disease mediated by regulatory B cells (Breg), which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient who needs it for a period of time sufficient for treatment The time of the disease of the Breg vector.

The present invention also provides a method for inhibiting the activity of regulatory T cells (Treg), which comprises contacting the regulatory T cells with an antibody that specifically binds to CD38.

The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSC), which comprises contacting the MDSC with an antibody that specifically binds to CD38.

The present invention also provides a method for inhibiting the activity of regulating B cells (Breg), which comprises contacting the Breg with an antibody that specifically binds to CD38.

The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of regulatory T cells (Treg) in the patient by administering an antibody that specifically binds to CD38 to the patient.

The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of bone marrow-derived suppressor cells (MDSC) in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method for treating a patient suffering from solid tumors, which comprises reducing the number of regulatory B cells (Breg) in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering an antibody that specifically binds to CD38 to the patient who needs it for a period of time sufficient to enhance the immune response.

In some embodiments, the patient has a viral infection.

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

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

In some embodiments, the Teff response is ^{mediated by CD4 +} T cells or CD8 ⁺ T cells.

In some embodiments, the Teff response is ^{mediated by CD4 +} T cells.

In some embodiments, the Teff response is ^{mediated by CD8 +} T cells.

In some embodiments, the Teff response is an increase in the number of ^{CD8 + T cells, an increase in CD8 +} T cell proliferation, an increase in T cell lineage expansion, an increase in CD8 ⁺ memory cell formation, and an increase in antigen-dependent antibody production. , Or increased production of cytokines, chemokines, or interleukins.

The proliferation of T cells can be achieved, for example, by measuring the rate of DNA synthesis using tritiated thymidine, or measuring the production of interferon-γ (IFN-γ) in vitro, or measuring the number of T cells in the cell population of patient samples using known methods. Evaluate in absolute numbers or percentages.

Homogenous expansion can be assessed by, for example, sequencing the TCR of the T cell pool using known methods.

ve)T細胞(CD45RO^-/CD62L⁺)對記憶T細胞(CD45RO⁺/CD62L^高)之比率來評估。 The formation of memory cells can be measured by using, for example, FACS to measure the initial (na

VE) T cells (CD45RO ^- / CD62L ⁺⁾ ratio of memory T cells (CD45RO ⁺ / CD62L ^high) of the assessed.

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. Production of IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, and IL-23, MIP-1α, MIP-1β, RANTES, CCL4) Use standard methods such as ELISA or ELLISPOT assay for evaluation.

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

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

Similarly, the meaning of "reduce (reducing)" or "decreasing (decrease)" the number of Treg, MDSC, and/or Breg is easy to understand. In a test sample or in a subject when compared to a control, for example, in a patient treated with an anti-CD38 antibody when compared to the same patient before treatment, or in response to an anti-CD38 antibody treatment When compared with those who did not respond to the same treatment or the group of patients, the reduction 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 more. Generally speaking, the reduction is statistically significant.

In some embodiments, antibodies that specifically bind to CD38 inhibit the function of immunosuppressive cells.

In some embodiments, the immunosuppressive cell line is regulatory T cells (Treg), bone marrow-derived suppressor cells (MDSC), or regulatory B cells (Breg).

In some embodiments, Tregs are CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cells.

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

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

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

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

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

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

In some embodiments, Treg killing is through the ADCC medium.

In some embodiments, CD38 ⁺ Treg is killed.

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

Since CD38 is only expressed in part of Treg and MDSC, it is expected that treatment of patients with solid tumors will not lead to systemic depletion of Treg and MDSC, and therefore may provide an improved safety profile.

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

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

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

In some embodiments, MDSC function is ^{inhibited by killing CD38 +} MDSC.

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

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

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

In some embodiments, Breg are CD19 ⁺ CD24 ⁺ CD38 ⁺ cells.

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

In some embodiments, Breg function is ^{inhibited by killing CD38 +} Breg.

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

In some embodiments, Breg killing is through the ADCC medium.

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

Treg plays a key role in maintaining tolerance of the peripheral body. The naturally occurring CD4 ⁺ CD25 ^hi Treg line is produced in the thymus and expresses Foxp3, which is a transcription factor required to establish and maintain Treg lineage identification and suppression functions. Tregs can accumulate in diseased sites (for example, in tumors), where they inhibit the effector function of tumor antigen-specific T cells, resulting in insufficient anti-tumor response. The increased density of tumor infiltrating Foxp3 ⁺ Treg is associated with poor prognosis in various solid tumors (including pancreatic cancer, ovarian cancer, and hepatocellular carcinoma). The depletion of Treg leads to enhanced anti-tumor immunity and tumor rejection in murine models, but it can also lead to the development of autoimmune diseases.

Bone marrow-derived suppressor cells (MDSC) are a heterogeneous group of early bone marrow precursor cells, immature granular spheres, macrophages, and dendritic cells at different stages of differentiation. They accumulate in large amounts in cancer patients, and they have powerful immunosuppressive functions, inhibiting the cytotoxic activity of both natural killer cells (NK) and natural killer T cells (NKT), and are ^{mediated by CD8 +} T cells The adaptive immune response. Although the mechanism of NK cell inhibition is currently not well understood, MDSC-mediated T cell inhibition is responsible for multiple pathways, including the production of arginase 1/ARG1 and the up-regulation of nitric oxide synthase 2 (NOS2) . ARG1 and NOS2 metabolize L-arginine, and together or separately block the translation of T cell CD3ζ chain, inhibit T cell proliferation, and promote T cell apoptosis. In addition, MDSC secretes immunosuppressive cytokines and induces regulatory T cell development.

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

MDSC has been described 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 Res Clin Oncol 136: 35-45; Ko et al. (2009) Clin Cancer Res 15: 2148-2157; Morse et al. (2009) ) Expert Opin Biol Ther 9:331-339; Diaz-Montero et al. (2009) Cancer Immunol Immunother 58:49-59; Corzo et al. (2009) J Immunol 182:5693-5701). Among cancer patients, Diaz et al. (Diaz-Montero et al. (2009) Cancer Immunol Immunother 58:49-59) proposed that the accumulation of MDSC is associated with more serious diseases and poor prognosis.

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

"Antibody-dependent cytotoxicity", "antibody-dependent cell-mediated cytotoxicity", or "ADCC" is a mechanism for inducing cell death, which depends on the target cells coated by the antibody and the effector cells with lytic activity (such as natural Killer cells, monocytes, macrophages, and neutrophils) interact via Fcγ receptors (FcγR) expressed on effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcvRIIIa. The death of antibody-coated target cells (such as CD38 expressing cells) occurs due to effector cell activity, which is through the secretion of membrane pore-forming proteins and proteases. In order to evaluate the ADCC activity of an antibody that specifically binds to CD38, the antibody can be added to a combination of CD38 expressing cells and immune effector cells, which can be activated by the antigen-antibody complex to cause cell lysis of target cells. Cell lysis is generally detected by the release of labels (such as radioactive substrates, fluorescent dyes, or natural intracellular proteins) from the lysed cells. Exemplary effector cells used in these assays include peripheral blood mononuclear cells (PBMC) and NK cells. Exemplary target cells include Tregs or MDSCs that express CD38. In an exemplary assay, target cells were ^{labeled with 20 μCi of 51} Cr for 2 hours and washed thoroughly. The cell concentration of these target cells can be adjusted to 1×10 ⁶ cells/ml, and various concentrations of anti-CD38 antibodies can be added. Start the measurement by adding target cells with an effect: target cell ratio of 40:1. After culturing at 37°C for 3 hours, the measurement was stopped by centrifugation, and the ⁵¹ Cr released from the lysed cells was measured in a scintillation counter. The percentage of cytotoxicity can be calculated as the maximum percentage of lysis that can be induced by adding 3% perchloric acid to the target cells.

"Antibody-dependent cellular phagocytosis"("ADCP") refers to a mechanism through internalization of phagocytes (such as macrophages or dendritic cells) to destroy antibody-coated target cells. ADCP can be assessed by using CD38-expressing Treg or MDSC as target cells (which are engineered to express GFP or other marker molecules). Effect: The target cell ratio can be, for example, 4:1. The effector cells and target cells can be cultured for 4 hours in the presence or absence of anti-CD38 antibodies. After culturing, the cells can be separated using a cell stripping solution (accutase). Macrophages can be identified by coupling with fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the percentage of phagocytosis can be determined based on the ^{% GFP fluorescence in these CD11 +} CD14 ⁺ macrophages using standard methods.

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

The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Human IgG1 or IgG3 is N-glycosylated at Asn297 and most of the glycans are in the well-known form of biantennary G0, G0F, G1, G1F, G2, or G2F. Antibodies produced by unengineered CHO cells usually have a glycan fucose content of at least 85%. Removal of core trehalose from biantennary complex oligosaccharides attached to the Fc region enhances the ADCC of antibodies by improving FcγRIIIa binding without changing antigen binding or CDC activity. These mAbs can be achieved using different methods that have been reported to cause the successful expression of relatively high amounts of defucosylated antibodies (Fc oligosaccharides with biantennary complex type), such as controlling the incubation osmotic pressure (Konno et al. , (2012) Cytotechnology 64: 249-65), using the variant CHO strain Lec13 as the host cell line (Shields et al., (2002) J Biol Chem 277: 26733-26740), using the variant CHO strain EB66 as the host cell line (Olivier et al., (2010) MAbs 2(4), Epub ahead of print; PMID: 20562582), using the rat fusion tumor cell line YB2/0 as the host cell line (Shinkawa et al., (2003) J Biol Chem 278 : 3466-3473), the introduction of a short interfering RNA specifically targeting α1,6-fucosyltrasferase (1,6-fucosyltrasferase, FUT8 ) gene (Mori et al. (2004) Biotechnol Bioeng 88:901-908), co-expression or β-1,4- N - acetyl glucosaminyl transferase III (β-1,4-N- acetylglucosaminyltransferase III) and the group's high α- mannosidase II (Golgi α-mannosidase II) or Keefe Kifunensine (a potent α-mannosidase I inhibitor) (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). The ADCC induced by the anti-CD38 antibody used in the method of the present invention and in some examples of each numbered example below can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 of the amino acid (residue numbering according to the EU index), such as U.S. Patent No. 6,737,056 As described in.

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

In some embodiments, the antibody that specifically binds CD38 contains a substitution (residue Numbering is based on EU index).

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

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

The substitution in Fc and the reduced trehalose content can enhance the ADCC activity of the antibody that specifically binds to CD38.

"Fucose content" means the amount of trehalose monosaccharide in the sugar chain at Asn297. The relative amount of trehalose is the percentage of trehalose-containing structures relative to all carbohydrate structures. These can be characterized and quantified by a variety of methods, such as: 1) Using samples treated with N-glycosidase F (such as complex, hybrid, and oligo-and high-mannose structures) MALD1-TOF, as described in International Patent Publication No. WO2008/077546; 2) Enzymatic release of Asn297 glycan, followed by derivatization and fluorescence detection by HPLC (UPLC) and/or HPLC-MS (UPLC-MS) MS) to detect/quantify; 3) Intact protein analysis of natural or reduced mAb, using Endo S or other glycans that cleave Asn297 glycans between the first and second GlcNAc monosaccharides, leaving behind those connected to the first GlcNAc Trehalose enzyme treatment or no treatment; 4) Use enzymatic digestion (such as trypsin or endopeptidase Lys-C) to digest mAb into constituent peptides, and then use HPLC-MS (UPLC -MS) separation, detection and quantification; or 5) specific enzymatic deglycosylation at Asn 297 with PNGase F to separate mAb oligosaccharides from mAb proteins. The released oligosaccharides can be labeled with fluorophores, separated and identified by a variety of complementary technologies, which allow: the comparison of experimental and theoretical quality by matrix-assisted laser desorption free (MALDI) mass spectrometry (MALDI) mass spectrometry Characterize the structure of glycans, determine the degree of sialylation by ion exchange HPLC (GlycoSep C), separate and quantify oligosaccharide forms by normal phase HPLC (GlycoSep N) according to hydrophilicity criteria, and by high efficiency Capillary electrophoresis-laser induced fluorescence (HPCE-LIF) separation and quantification of oligosaccharides.

As used herein, "low fucose" or "low fucose content" means that the trehalose content of the antibody is about 0% to 15%.

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

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

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

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

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

In some embodiments, antibodies that specifically bind to CD38 increase ^{the ratio of CD8 +} T cells to Treg.

In some embodiments, antibodies that specifically bind to CD38 increase the ratio of ^CD8+ central memory cells to CD8 ^{+ naive cells.} CD8 ⁺ central memory cells can be identified as CD45RO ⁺ /CD62L ^{+ tall} cells. CD8 ⁺ naive cells can be identified as CD45RO-/CD62L ⁺ cells.

In some embodiments, the antibodies that specifically bind to CD38 are non-agonistic antibodies.

A non-agonist anti-acting system that specifically binds to CD38 refers to a system that does not induce significant proliferation of peripheral blood mononuclear cell samples in vitro when compared to the proliferation induced by isotype control antibodies or only mediators after binding to CD38 Antibody.

In some embodiments, non-agonistic antibodies that specifically bind to CD38 induce the proliferation of peripheral blood mononuclear cells (PBMC) in a statistically insignificant manner. PBMC proliferation can be assessed by culturing PBMC isolated from healthy donors in 200 μl RPMI with 1×10 ⁵ cells/well in a flat-bottomed 96-well plate in the presence or absence of the test antibody. After culturing at 37°C for 4 days, 30 μl of ³ H-thymidine (16.7 μCi/ml) can be added, and the incubation can be continued overnight. ³ H-thymidine incorporation can be evaluated using Packard Cobra gamma counter (Packard Instruments, Meriden, DT, USA) according to the manufacturer's instructions. The data can be calculated as the average cpm (±SEM) of PBMC obtained from several donors. The statistical significance or insignificance between samples grown in the presence or absence of the test antibody is calculated using standard methods.

^{An exemplary anti-CD38 anti-system DARZALEX(TM)} (dalazumab) that can be used in the method of the present invention. DARZALEX ^TM (dalatumumab) includes the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences shown in SEQ ID NOs: 4 and 5, respectively, which are respectively SEQ ID NO: 6, The heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 of 7, and 8, and the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 of SEQ ID NOs: 9, 10, and 11, respectively, and It is of the IgG1/κ subtype and is described in U.S. Patent No. 7,829,693. The heavy chain amino acid sequence of DARZALEX ^(TM) (dalazumab) is shown in SEQ ID NO: 12, and the light chain amino acid sequence is shown in SEQ ID NO: 13.

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

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

SEQ ID NO: 1

SEQ ID NO: 2 SKRNIQFSCKNIYR

SEQ ID NO: 3 EKVQTLEAWVIHGG

SEQ ID NO: 4

SEQ ID NO: 5

SEQ ID NO: 6 SFAMS

SEQ ID NO: 7 AISGSGGGTYYADSVKG

SEQ ID NO: 8 DKILWFGEPVFDY

SEQ ID NO: 9 RASQSVSSYLA

SEQ ID NO: 10 DASNRAT

SEQ ID NO: 11 QQRSNWPPTF

SEQ ID NO: 12

SEQ ID NO: 13

A well-known in vitro method can be used to evaluate the competition of the binding of an antibody to CD38 ^{with a reference antibody (such as DARZALEX(TM} ) with VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5). In an exemplary method, the recombinant CD38-expressing CHO cells can be incubated with an unlabeled reference antibody for 15 minutes at 4°C, and then incubated with an excess of fluorescently labeled test antibody at 4°C for 45 minutes. After washing in PBS/BSA, standard methods can be used to measure fluorescence by flow cytometry. In another exemplary method, the extracellular portion of human CD38 can be coated on the surface of an ELISA dish. An excess of unlabeled reference antibody can be added for about 15 minutes, and then the biotinylated test antibody can be added. After washing in PBS/Tween, use streptavidin (horseradish peroxidase, HRP) to detect the binding of the test biotinylated antibody and use standard methods to detect the signal . In these competition assays, it is obvious that the reference antibody can be labeled and the test antibody can be unlabeled. When the reference antibody inhibits the binding of the test antibody to CD38, or the test antibody inhibits 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 test antibody competes with the reference antibody. The epitope of the test antibody can be further defined by, for example, peptide mapping or hydrogen/deuterium protection assay using known methods, or by crystal structure determination.

Antibodies that bind to the SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) regions of human CD38 (SEQ ID NO: 1) can be produced, for example, by the following: using standard methods and the methods described herein Mice were immunized with peptides having the amino acid sequences shown in SEQ ID NOs: 2 and 3, and the resulting antibodies bound to the peptides were qualitatively used, for example, ELISA or mutagenesis studies.

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

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

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

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

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

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

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

Another exemplary anti-CD38 antibody system that can be used in any embodiment of the present invention: mAb003, which contains the VH and VL sequences of SEQ ID NOs: 14 and 15, respectively, and is described in US Patent No. 7,829,693. The VH and the VL of mAb003 can be expressed as IgG1/κ.

SEQ ID NO: 14

mAb024，其包含分別為SEQ ID NO：16及17的VH及VL序列並描述於美國專利第7,829,693號中。mAb024的該VH及該VL可被表示為IgG1/κ。 SEQ ID NO: 15

mAb024, which includes the VH and VL sequences of SEQ ID NOs: 16 and 17, respectively, and is described in US Patent No. 7,829,693. The VH and the VL of mAb024 can be expressed as IgG1/κ.

SEQ ID NO: 16

SEQ ID NO: 17

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

SEQ ID NO: 18

伊沙妥昔單抗(Isatuximab)；其包含分別為SEQ ID NO：20及21的VH及VL序列並描述於美國專利第8,153,765號中。伊沙妥昔單抗之VH及VL可被表示為IgG1/κ。 SEQ ID NO: 19

Isartuximab (Isatuximab); it contains the VH and VL sequences of SEQ ID NOs: 20 and 21, respectively, and is described in US Patent No. 8,153,765. The VH and VL of Isartuximab can be expressed as IgG1/κ.

SEQ ID NO 20:

SEQ ID NO: 21:

Other exemplary anti-CD38 antibodies that can be used in the methods of the present invention include those described in International Patent Publication No. WO05/103083, International Patent Publication No. WO06/125640, International Patent Publication No. WO07/042309, International Patent Publication No. WO08/047242, or International Patent Publication No. WO14/178820.

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

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

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

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

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

In some embodiments, the solid tumor is melanoma.

In some embodiments, the solid tumor is lung cancer.

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

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

In some embodiments, the solid tumor is lung adenocarcinoma.

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

In some embodiments, the solid tumor is mesothelioma.

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

In some embodiments, the solid tumor is colorectal cancer.

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

In some embodiments, the solid tumor is stomach cancer.

In some embodiments, the solid tumor is ovarian cancer.

In some embodiments, the solid tumor is gastric cancer.

In some embodiments, the solid tumor is liver cancer.

In some embodiments, the solid tumor is pancreatic cancer.

In some embodiments, the solid tumor is thyroid cancer.

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

In some embodiments, the solid tumor is a cancer of the esophagus or gastrointestinal tract.

In some embodiments, the solid tumor is breast cancer.

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

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

In some embodiments, the solid tumor is urethral cancer.

In some embodiments, the solid tumor is urogenital cancer.

In some embodiments, the solid tumor is endometriosis.

In some embodiments, the solid tumor is cervical cancer.

In some embodiments, solid tumors are metastatic lesions of cancer.

In some embodiments, solid tumors lack detectable CD38 manifestations.

When compared to a control, for example, the expression detected with an anti-CD38 antibody using a well-known method is compared with the expression detected with an isotype control antibody, the expression of CD38 in solid tumor tissue or on cells isolated from the solid tumor is statistically When insignificant, solid tumors lack detectable CD38 manifestations.

The anti-CD38 antibody used in the method of the present invention can also be re-selected from, for example, a phage display library, where the phage system is engineered to express human immunoglobulin or parts 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, a phage display library (expressing antibody heavy and light chain variable regions) as a fusion protein with a phage pIX coat protein, as described in Shi et al., (2010) J Mol Biol 397: 385-96 and International Patent Publication No. WO09/085462. The antibody library can be screened for binding to the extracellular domain of human CD38, and the obtained positive clones are further characterized. The lysate from the clones isolates the Fab, and is subsequently selected as a full-length antibody. This phage display method for isolating human antibodies has been established in the art. See for example: U.S. Patent No. 5,223,409, U.S. Patent No. 5,403,484, U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,885,793 , U.S. Patent No. 6,521,404, U.S. Patent No. 6,544,731, U.S. Patent No. 6,555,313, U.S. Patent No. 6,582,915, and U.S. Patent No. 6,593,081.

In some embodiments, the anti-CD38 antibody system IgG1, IgG2, IgG3, or IgG4 isotype.

The Fc part of the antibody can mediate the effector function of the antibody, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). This function can be mediated by the binding of Fc effector domains to Fc receptors on immune cells with phagocytic or lytic activity, or by the binding of Fc effector domains to components of the complement system. Generally, the effects mediated by cells or complement components that bind to Fc will result in the inhibition or depletion of target cells (for example, CD38 expressing cells). Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 show differential ability in effector functions. ADCC can be mediated by IgG1 and IgG3, ADCP can be mediated by IgG1, IgG2, IgG3, and IgG4, and CDC can be mediated by IgG1 and IgG3.

An antibody that is substantially the same as the antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 can be used in the method of the present invention. The term "substantially identical" as used herein means that the VH or VL amino acid sequences of the two antibodies compared are identical or have "insubstantial differences". The insubstantial difference is that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 in the heavy or light chain of the antibody will not adversely affect the performance of the antibody Affected amino acid substitution. The identity percentage can be determined, for example, by using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA) to make a comparison. The protein sequence of the present invention can be used as a query sequence to perform searches against public or patent databases to, for example, identify related sequences. Exemplary programs used to perform these searches are XBLAST or BLASTP programs (http_//www_ncbi_nlm/nih_gov), or GenomeQuest ^TM (GenomeQuest, Westborough, MA) package with default settings. Exemplary substitutions that can be made to an antibody that specifically binds to CD38 are, for example, conservative substitutions with amino acids with similar charge, hydrophobicity, or stereochemical properties. Conservative substitutions can also be made to improve antibody properties (such as stability or affinity), or to improve the effector function of the antibody. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made in the heavy or light chain of the anti-CD38 antibody . In addition, any natural residues in the VH or VL can also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55- 67, 1998; Sasaki et al., Adv Biophys 35: 1-24, 1998). The desired amino acid substitution can be determined by a person with ordinary knowledge in the field when these substitutions are desired. Amino acid substitution can be performed, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). The library of variants can be generated using well-known methods, such as using random (NNK) or non-random codons (such as DVK codons), which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp), and then screen the library of variants to find variants with desired properties. The generated variants can be tested in vitro for their binding to CD38, their ability to induce ADCC, ADCP, or apoptosis, or to regulate CD38 enzyme activity using the methods described herein.

In some embodiments, antibodies that specifically bind to CD38 can bind to human CD38 with _{a range of affinities (K D ).} In an embodiment according to the present 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, K _D is equal to or less than about 10 ^-7 M, such as But not limited to, 1 to 9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 ^-8 M, 10 ^-9 M, 10 ^-10 M , 10 ^-11 M, 10 ^-12 M, 10 ^-13 M, 10 ^-14 M, 10 ^-15 M, or any range or value thereof, as determined by surface plasmon resonance or Kinexa method, as in the technical field Practiced by those with ordinary knowledge. An exemplary affinity is equal to or less than 1×10 ^-8 M. Another exemplary affinity is equal to or less than 1×10 ⁻⁹ M.

In some embodiments, the anti-system bispecific antibody that specifically binds to CD38. The VL and/or VH regions of existing anti-CD38 antibodies or re-identification as described herein The VL and VH regions can be engineered into bispecific full-length antibodies. These bispecific antibodies can be produced 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 document: US Patent No. 7,695,936 ; International Patent Publication No. WO04/111233; U.S. Patent Publication No. US2010/0015133; U.S. Patent Publication No. US2007/0287170; International Patent Publication No. WO2008/119353; U.S. Patent Publication No. US2009/0182127; U.S. Patent Publication No. US2010/0286374; Patent Publication No. US2011/0123532; International Patent Publication No. WO2011/131746; International Patent Publication No. WO2011/143545; or US Patent Publication No. US2012/0149876. Other bispecific structures such as dual variable domain immunoglobulins (International Patent Publication No. WO2009/134776) that can incorporate the VL and/or VH regions of the antibody of the present invention, or include various dimerization domains to link with different specificities The structure of two antibody arms, such as leucine zipper or collagen dimerization domain (International Patent Publication No. WO2012/022811, U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441).

For example, bispecific antibodies can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies, and under reducing conditions (to allow the disulfide bond Isomerization) to form the bispecific heterodimeric antibody with two parent monospecific homodimeric antibodies according to the method described in International Patent Publication No. WO2011/131746. In these methods, the first monospecific bivalent antibody (eg, anti-CD38 antibody) and the second monospecific bivalent antibody system are engineered to have certain substitutions in the CH3 domain that promote heterodimer stability ; These antibodies are cultured together under reducing conditions sufficient to allow the cysteine in the hinge region to carry out disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The culture conditions can be optimally restored to non-reducing. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, ginseng (2- Carboxyethyl) phosphine (TCEP), L-cysteine and β-mercaptoethanol. The reducing agent is preferably selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and ginseng (2 -Carboxyethyl) phosphine. For example, it can be used 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 Exist and incubate at a pH of 5 to 8 (for example, at a pH of 7.0 or at a pH of 7.4) for at least 90 minutes.

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

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

Administration/medical composition

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

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

Antibodies that specifically bind to CD38 can be administered to the patient by any suitable route, such as parentally administered (by intravenous (iv) infusion or bolus injection), intramuscular Or subcutaneously or intraperitoneally. iv infusion can be given within, for example, 15, 30, 60, 90, 120, 180, 240 minutes, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours .

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

A fixed unit dose may also be given, such as 50, 100, 200, 500, or 1000 mg, or the dose may be based on the surface area of the patient, such as 500, 400, 300, 250, 200, or 100 mg/m ² . Often doses between 1 and 8 times (for example, 1, 2, 3, 4, 5, 6, 7, or 8 times) can be administered, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses.

The antibody that specifically binds to CD38 in the method of the present invention can be administered in one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, and six weeks. , Seven weeks, two months, three months, four months, five months, six months, or more later. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, the antibody that specifically binds to CD38 in the method of the present invention can be administered at 8 mg/kg or at 16 mg/kg at weekly intervals for 8 weeks, and then at 8 mg/kg or at 16 mg/kg every two weeks. The administration continued for another 16 weeks, followed by intravenous infusion at 8 mg/kg or 16 mg/kg every four weeks.

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

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

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

The antibody that specifically binds to CD38 in the method of the present invention can be lyophilized for storage, and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional protein preparations and well-known freeze-drying and reconstitution techniques can be used.

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

In the method of the present invention, the antibody that specifically binds to CD38 can be administered together with any one or more of chemotherapeutic drugs or other anti-cancer therapeutic agents known to those with ordinary knowledge in the art. Chemotherapeutic agents are chemical compounds that can be used to treat cancer, and include growth inhibitors or other cytotoxic agents, and include alkylating agents, antimetabolites, anti-microtubule inhibitors, and topoisomerase inhibitors , Receptor tyrosine kinase inhibitors, angiogenesis inhibitors, and the like. Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); sulfonic acid alkyl esters, such as busulfan (busulfan), improsulfan (improsulfan), And piposulfan (piposulfan); aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; Ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylene thiophosphoramide ( triethylenethiophosphaoramide, and trimethylolomelamine; nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine (estramustine), ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin ), cholesterol phenesterine (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); nitrosureas, such as carmus Carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as Aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, cactinomycin (Calicheamicin), carabicin (carabicin), carminomycin (carminomycin), oncinomycin (c arzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-n-leucine Acid, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin ), mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, triiron Adriamycin (quelamycin), rhodoubicin (rodorubicin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), netstatin (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate and 5-FU; folate analogs, such as denopterin, methotrexate, pterorin ( pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs , Such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine ), doxifluridine, enocitabine, floxuridine; androgen, such as calusterone, dromostanolone propionate, sulfide Androstanol (epitiostanol), mepitiostane (mepitiostane), testolactone (testolactone); anti-adrenal agents, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); Supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; betabuxin (bestrabucil); bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; Elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone ( mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid ; 2-ethylhydrazide (2-ethylhydrazide); procarbazine; PSK®; razoxane; sizofiran; spirogermanium; Alternaria tenuifonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2"-trichlorotriethylamine;carbamate; vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard ( mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); ring Phosphasamide; thiotepa; taxol-like or members of the taxane family, such as paclitaxel (TAXOL®), docetaxel (TAXOTERE®), and its analogs; chlorambucil ); gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; Etoposi de,VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; navelbine Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; receptor tyrosine kinase inhibition Agents and/or angiogenesis inhibitors, including NEXAVAR® (sorafenib (sorafenib)), SUTENT® (sunitinib), VOTRIENT ^TM (pazopanib), PALLADIA ^TM (support Toceranib), ZACTIMA ^TM (vandetanib), RECENTIN® (cediranib), regorafenib (BAY 73-4506), axitinib (axitinib) (AG013736), Lestaurtinib (CEP-701), TARCEVA® (erlotinib), IRESSA ^TM (gefitinib), Gilotrif ^® (Afatinib) Afatinib), TYKERB® (lapatinib), neratinib, and the like, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. This definition also includes antihormonal agents used to modulate or inhibit the effect of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5) -Imidazole, 4-hydroxy tamoxifen, trioxifene (trioxifene), raloxifene (keoxifene), LY 117018, onapristone, and FARESTON® (toremifene) ; And antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and the above Any pharmaceutically acceptable salt, acid, or derivative. For example, other conventional cytotoxic compounds disclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing may also be suitable for the method of the present invention.

Exemplary agents that can be used in combination with antibodies that specifically bind to CD38 in the method of the present invention include tyrosine kinase inhibitors and targeted anti-cancer therapies, such as IRESSA ^TM (gefitinib) and Tarceva ^® (erlotinib) Ni), and other antagonists of HER2, HER3, HER4, or VEGF. Exemplary HER2 antagonists include CP-724-714, HERCEPTIN ^TM (trastuzumab (trastuzumab)), OMNITARG ^TM (pertuzumab (pertuzumab)), TAK-165 , TYKERB® ( lapatinib) (EGFR and HER2 inhibitors), and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies (see, for example, U.S. Patent Publication No. 2004/0197332). Exemplary HER4 antagonists include anti-HER4 siRNA (see, eg, Maatta et al., Mol Biol Cell 17: 67-79, 2006). An exemplary VEGF antagonist is Avastin ^™ (bevacizumab).

Exemplary agents that can be used in combination with an antibody that specifically binds to CD38 in the method of the present invention include standard care drugs for solid tumors, or immune checkpoint inhibitors.

The second therapeutic agent in the method of the present invention may be an immune checkpoint inhibitor.

In some embodiments, 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.

In some embodiments, 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.

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

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

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

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

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

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

Any antagonistic anti-PD-1 antibody can be used in the method of the invention. Exemplary anti-PD-1 antibodies that can be used OPVIDO® (nivoluzumab) and KEYTRUDA® (pelizumab). OPVIDO® (nivolumab) is described in, for example, US Patent No. 8,008,449 (antibody 5C4) and includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25. KEYTRUDA® (Peclizumab) is described in, for example, US Patent No. 8,354,509 and includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23. The amino acid sequence of Nivolumab and Peclizumab can also be obtained through CAS registration. Additional PD-1 anti-systems that can be used are described in U.S. Patent No. 7,332,582, U.S. Patent Publication No. 2014/0044738, International Patent Publication No. WO2014/17966, and U.S. Patent Publication No. 2014/0356363.

"Antagonist" refers to a molecule that, when bound to a cellular protein, inhibits the reaction or activity induced by at least one of the protein's natural ligands. A molecule, when at least one reaction or activity is inhibited by at least about 30%, 40%, 45%, 50% more than the at least one reaction or activity in the absence of an antagonist (eg, negative control) , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or when compared to inhibition in the absence of an antagonist, inhibit When it is statistically significant, it is 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 receptor PD-L1 or PD-L2 can be ^{the reduction of antigen-specific CD4 +} or CD8 ⁺ cell proliferation or the interferon-γ (IFN-γ (IFN- γ) Reduction in production, which leads to suppression of immune responses against, for example, tumors. The typical response or activity induced by the binding of TIM-3 to its receptor (such as galectin-9) can be ^{the reduction of antigen-specific CD4 +} or CD8 ⁺ cell proliferation, and the reduction of IFN-γ production by T cells , Or ^{a reduction in the surface expression of CD137 on CD4+} or CD8 ⁺ cells, which results in suppression of 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 the inhibitor pathway.

SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO: 24

SEQ ID NO: 25

Anti-PD-L1 antibodies that enhance immune response can be used in the methods of the present invention (for example, antagonistic anti-PD-L1 antibodies). Exemplary anti-PD-L1 antibody systems that can be used, durvalumab, atezolizumab, and avelumab, and are described in, for example, U.S. Patent Publication No. 2009/0055944 , U.S. Patent No. 8,552,154, U.S. Patent No. 8,217,149, and U.S. Patent No. 8,779,108.

Devalumumab includes the VH of SEQ ID NO:26 and the VL of SEQ ID NO:27.

Atezizumab includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29.

Aveluzumab includes the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31.

SEQ ID NO: 26

SEQ ID NO: 27

SEQ ID NO: 28

SEQ ID NO: 29

SEQ ID NO: 30

SEQ ID NO: 31

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 to the patient who needs it. The combination of the body (which includes the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29) is sufficient for a period of time The time to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-1 antibody (which includes the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 25) are combined for a period of time sufficient to enhance the immune response.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-1 antibody (which includes the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23) are combined for a period of time sufficient to enhance the immune response.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27) are combined for a period of time sufficient to enhance the immune response.

The present invention also provides a method for enhancing the immune response of a patient, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH and SEQ ID of SEQ ID NO: 4 to the patient who needs it. NO: 5 VL) and anti-PD-L1 antibody (which includes the VH of SEQ ID NO: 28 and the VL of SEQ ID NO: 29) are combined for a period of time sufficient to enhance the immune response.

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

The present invention also provides a method for treating a patient suffering from colorectal cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PID-1 antibody to the patient in need For a period of time sufficient to treat the colorectal cancer.

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

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

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

The present invention also provides a method for treating a patient suffering from lung cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L1 antibody to the patient who needs it for a period of time Enough time to treat this lung cancer.

The present invention also provides a method for treating a patient suffering from lung cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L2 antibody to the patient who needs it for a period of time Enough time to treat this lung cancer.

The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-1 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.

The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L1 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.

The present invention also provides a method for treating a patient suffering from prostate cancer, which comprises administering a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonist anti-PD-L2 antibody to the patient who needs it. A period of time sufficient to treat the prostate cancer.

Anti-LAG-3 antibodies that enhance immune response can be used in the methods of the present invention. Exemplary anti-LAG-3 resistance systems that can be used are described in, for example, International Patent Publication No. WO2010/019570.

Anti-CTLA-4 antibodies that enhance immune response can be used in the methods of the present invention. An exemplary anti-CTLA-4 antibody system that can be used Ipilimumab.

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

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

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

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

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

SEQ ID NO: 32

SEQ ID NO: 33

SEQ ID NO: 34

SEQ ID NO: 35

SEQ ID NO: 36

SEQ ID NO: 37

SEQ ID NO: 38

SEQ ID NO: 39

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-PD-1 antibody (which includes the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-TIM-3 antibody (which includes the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37) is combined for a period of time sufficient to treat the solid tumor.

The present invention also provides a method for treating patients suffering from solid tumors, which comprises administering a therapeutically effective amount of an antibody that specifically binds to CD38 (which includes the VH of SEQ ID NO: 4 and The combination of the VL of SEQ ID NO: 5) and the anti-TIM-3 antibody (which includes the VH of SEQ ID NO: 38 and the VL of SEQ ID NO: 39) for a period of time sufficient to treat the solid tumor.

In the method of the present invention, the combination of an antibody that specifically binds to CD38 and a second therapeutic agent can be administered within any conventional time period. For example, the antibody that specifically binds to CD38 and the second therapeutic agent can be administered to the patient on the same day and even by the same intravenous infusion. However, the antibody that specifically binds to CD38 and the second therapeutic agent can also be administered on alternate days, alternate weeks or months, and so on. In some methods, the antibody that specifically binds to CD38 and the second therapeutic agent can be administered at a sufficiently close time so that they are present at a detectable level (for example, in serum) in the treated patient. middle. In some methods, the entire course of treatment with an antibody that specifically binds to CD38 (which consists of several doses over a period of time) is before or after the course of treatment with the second therapeutic agent (which consists of several doses). Rear. A recovery period of 1, 2, or several days or weeks can be used between the administration of the antibody that specifically binds to CD38 and the second therapeutic agent.

The combination of an antibody that specifically binds to CD38 or an antibody that specifically binds to CD38 and a second therapeutic agent can be combined with any form of radiation therapy (including external beam radiation, intensity-modulated radiation therapy (IMRT), focused radiation, and any Forms of radiosurgery (including Gamma Knife, Cyberknife, Linac, and Interstitial Radiation (e.g. implanted radioactive seeds, GliaSite balloon)), and/or surgery Apply together.

The available focused radiation methods include stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy (IMRT). It is obvious that stereotactic radiosurgery involves precise delivery of radiation to tumor tissues (such as brain tumors) while avoiding surrounding non-tumor, normal tissues. The radiation dose applied using stereotactic radiosurgery may vary, usually from 1 Gy to about 30 Gy, and may include intermediate ranges, including, for example, doses ranging from 1 to 5, 10, 15, 20, 25, and up to 30 Gy. Because of the non-invasive fixation device, stereotactic radiation does not need to be delivered in a single treatment. The treatment plan can be reliably repeated day after day, thereby allowing multiple fractional radiation doses to be delivered. When used to treat tumors over a period of time, the radiosurgery is called "fractionated stereotactic radiosurgery" or FSR. In contrast, stereotactic radiosurgery refers to one-session treatment. Fractionated stereotactic radiosurgery may result in a high treatment rate, that is, a high tumor cell killing rate and low impact on normal tissues. Tumors and normal tissues respond differently to high single-dose radiation than multiple low-dose radiation. A single large dose of radiation may kill more normal tissues than several small doses of radiation. Therefore, multiple small doses of radiation can kill more tumor cells without harming normal tissues. The radiation dose applied using fractionated stereotactic radiation may vary from 1 Gy to about 50 Gy, and may include intermediate ranges, including, for example, 1 to 5, 10, 15, 20, 25, 30, 40, and up to 50 Gy. Low divided dose. Intensity-modulated radiation therapy (IMRT) can also be used. IMRT is an advanced mode of high-precision three-dimensional conformal radiotherapy (3DCRT), which uses a computer-controlled linear accelerator to deliver precise radiation doses to malignant tumors or specific areas within the tumor. In 3DCRT, a multi-leaf collimator (MLC) is used to shape the contour of each radiation beam into a contour suitable for the target (from beam perspective (beam's eye view, BEV) point of view), thereby generating several beams. IMRT adjusts the intensity of the radiation beam in multiple small amounts, so that the radiation dose can more accurately conform to the three-dimensional (3-D) shape of the tumor. Therefore, IMRT enables higher radiation doses to be concentrated to the area within the tumor, while minimizing the dose to the surrounding normal key structures. IMRT improves the ability to adapt the treatment volume to the shape of a concave tumor, for example, when the tumor is entangled in fragile structures such as the spinal cord or major organs or blood vessels.

Subcutaneous administration of a pharmaceutical composition containing an antibody that specifically binds to CD38 and hyaluronidase

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

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

The pharmaceutical composition administered subcutaneously may contain between about 1,200 mg to 1,800 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition administered subcutaneously may contain about 1,200 mg of an antibody that specifically binds to CD38.

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

The pharmaceutical composition administered subcutaneously may contain about 1,800 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition administered subcutaneously may contain between about 30,000 U and 45,000 U of hyaluronidase.

The pharmaceutical composition administered subcutaneously may include about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

The subcutaneously administered pharmaceutical composition may include about 1,800 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

The pharmaceutical composition administered subcutaneously may include about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

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

The pharmaceutical composition for subcutaneous administration may include hyaluronidase rHuPH20, which has the amino acid sequence of SEQ ID NO:40.

rHuPH20 is a recombinant hyaluronidase (HYLENEX® recombinant) and is described in International Patent Publication No. WO2004/078140.

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.

SEQ ID NO: 40

The pharmaceutical composition containing the antibody that specifically binds to CD38 and hyaluronidase can be administered on one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, and six days. Repeat this after a week, seven weeks, two months, three months, four months, five months, six months, or longer. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, a pharmaceutical composition comprising an antibody that specifically binds to CD38 and hyaluronidase can be administered once a week for eight weeks, then once every two weeks for 16 weeks, and then once every four weeks. The pharmaceutical composition to be administered may include 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 pharmaceutical composition is about 20 mg/ml. The pharmaceutical composition to be administered may contain about 1,800 mg of specifically binding CD38 The antibody and about 45,000U of hyaluronidase. The pharmaceutical composition to be administered may include 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 include about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

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

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

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

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

Example 1. General materials and methods Sample collection and processing

The peripheral blood and bone marrow aspirates were collected in a heparinized tube at the base period immediately before the first infusion and at the specified time point during the treatment period. Most of the samples are evaluated using instant flow cytometry when they arrive at the central laboratory 24 to 48 hours after collection. Peripheral blood mononuclear cells (PBMC) were obtained from whole blood, isolated by density gradient centrifugation, and stored frozen until analysis. For the determination of T cell activation, homology, and CD38 ⁺ Treg inhibition, frozen PBMC samples are used, and samples before and after treatment are analyzed at the same time.

In the BARC global central laboratory (BARC global central laboratory), these samples are analyzed by flow cytometry, using pre-validated immunophenotyping assays to evaluate NK cells, T cells, B cells, myeloma cells (CD138 ⁺ ), And CD38 performance. In short, the blood samples and bone marrow samples were stained with the following multi-fluorescent dye antibody groups: Cell lineage group: PerCPCy5.5α-CD19 (purified 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 cell (T _reg ) group: APCα-CD25 (2A3; BD), PEα- CD127 (HIL-7R-M21; BD), APC-H7α-HLA-DR (G46-6; BD), and PerCPα-CD4 (L200; BD); naive/memory T cell group: APC-H7α- CD4 (RPA-T4; BD), PerCP-Cy5.5α-CD8 (RPA-T4 BD), PEα-CD62L (SK11; BD), and APCα-CD45RA (HI100; BD). CD38 expression was evaluated using Alexa 647-labeled antibody mAb 003, which is described in US Patent No. 7,829,693 and has the VH and VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15. The blood samples are prepared using different lysis-washing methods. Various antibodies are used for membrane or intracellular staining of bone marrow aspirate samples. Becton Dickinson FACS lysis solution is used to lyse red blood cells in surrounding blood samples, and Fix and Perm cell permeation reagents from Invitrogen are used for intracellular staining of bone marrow aspirate samples. The stained samples were obtained on the FACS Canto II flow cytometer, and the data was analyzed using FacsDiva software. Determine the absolute count of the immune cell population in the blood sample and the percentage of lymphocytes in the bone marrow sample at all time points tested.

T cell receptor (TCR) sequencing

T cell diversity is analyzed by using the genomic DNA of PBMC samples to perform deep sequencing of TCR rearrangement to assess CD8 ⁺ T cell homology. TCR sequencing was performed using the Immunoseq ^TM assay commercially available from Adaptive Biotechnologies, and the analysis was performed using a pre-qualified multiple polymerase chain reaction (PCR) assay (TR2015CRO-V-019), which was performed by a forward primer and a reverse primer As a result, these forward primers and reverse primers directly target the family of variable (V) genes (forward primers) and junction (J) genes (reverse primers). The primers of the V and J genes are used as a priming pair to amplify the recombined TCR of somatic cells, and each primer contains a specific universal DNA sequence. After the initial PCR amplification, each amplification was amplified a second time with forward primers and reverse primers. These forward primers and reverse primers contained universal sequences and adapter sequences required for Illumina DNA sequencing (adaptor sequence).

T cell response to viral antigens and allogeneic antigens

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

Regulating T cell (Treg) inhibition of effector cell function: Carboxyfluorescein succinimidyl ester (CFSE) dilution assay

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

Myelo-derived suppressor cell (MDSC) phenotype and DARZALEX ^TM (Dalazumab) ADCC of the medium

PBMC from three normal healthy donors were incubated with myeloma cell lines (RPMI8226, U266, H929) for six days, and the production of granular spherical MDSC (G-MDSC) (CD11b ⁺ CD14 ^- HLA ^- DR ^- CD15 ⁺⁾ CD33 ⁺ ), as described in Gorgun et al., Blood 121:2975-87, 2013. G-MDSC is not present in normal healthy PBMC, but after co-cultivation with all three myeloma cell lines, G-MDSC is present in 5 to 25% of the total PBMC population (data not shown). The gating strategy for flow cytometric evaluation of G-MDSC includes CD11b ⁺ as the first circle selection, followed by CD14 ^- and HLA ^- DR ^- circle selection, and then CD15 ⁺ and CD33 ⁺ circle selection. G-MDSC was cell sorted and evaluated CD38 expression level and sensitivity to ADCC mediated by ^{DARZALEX TM (dalazumab).} In order to evaluate the effect of DARZALEX ^TM (dalazumab) on ADCC/CDC of MDSC, serum containing complement or isotype control was added to the ADCC assay.

Initial and memory T cell analysis

Heparinized peripheral blood samples were obtained from the patients, and then each was infused with DARZALEX ^TM (dalazumab). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation, and stored in cryopreservation medium in liquid nitrogen (RPMI supplemented with 10% human serum and 10% dimethylsulfoxide) . For FACS analysis, PBMC were thawed, and 2×10 ⁶ cells/group were resuspended in phosphate buffered saline (PBS) with 0.05% azide and 0.1% HAS.

data analysis

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

Different statistical comparisons include: (i) the baseline level between responders and non-responders, (ii) the baseline level of responders and non-responders during treatment, and (iii) the baseline level between responders and non-responders Percentage change, (iv) The ratio of base period to treatment change. The comparisons first include the normality test using the Shapiro-Wilk test (Royston (1995) Remark AS R94: A remark on Algorithm AS 181: The W test for normality. Applied Statistics, 44, 547-551). It is found that almost all data does not have a normal distribution. The difference level test includes the non-parametric Wilcox rank sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons. Pages 27-33 (single sample), 68 -75 (double sample)) and t test after Box Cox conversion (Weisberg, S. (2014) Applied Linear Regression, Fourth Edition, Wiley Wiley, Chapter 7). For Box-Carks conversion, the decimal (1e-07) is added to a value equal to 0. In all cases, the two checks agree. Unless otherwise instructed, Wil Carson's grades and verified p-values are shown in tables throughout this manual. When testing the difference in treatment between responders and non-responders, each subject undergoes a two-group paired test, and in all other cases, a two-group unpaired test.

Because samples of various lymphocyte populations were not obtained at the same time point for different dosing schedules, the population modeling was carried out. Model fitting is performed on the level of visits. Population modeling of the population and activated NK cells involves fitting a broken stick model (Lutz et al., "Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships, implemented for mutagenicity data." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678.2(2009):118-122.). A linear mixed-effect model with random intercept and slope was fitted to the data of B cells, T cell subpopulations, and leukocytes, monocytes, neutrophils, and lymphocytic disease populations (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 mixed modeling is performed on the relative days after the start of treatment (ADY). This linear mixed model fitting system is performed on the logarithmic transformed response variable. In the case where the response variable value is equal to 0, 0.1 is added to all response variable values to allow modeling on a logarithmic scale.

Example 2. Research 54767414MMY2002 design (SIRIUS)

Study 54767414MMY2002 (SIRIUS) target group of patients with advanced multiple myeloma, who have received at least 3 therapies before, including proteasome inhibitors (PI) and immunomodulatory drugs (IMiD), or the like PI and IMiD are double intractable. The primary endpoint/final analysis of the response evaluation is based on the independent review committee (IRC) and computerized algorithm evaluation, which uses the 2011 IMWG criteria (clinical study report: an open-label, multi-center, phase 2 trial , Which explores the efficacy and safety of DARZALEXTM (dalazumab) in subjects with multiple myeloma who have previously received at least 3 therapies (including proteasome inhibitors and IMiD) or those subjects are Proteasome inhibitors and IMiD are double refractory (EDMS-ERI-92399922; de Weers et al. (2011) J Immunol 186(3): 1840-1848)).

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

In this study, a total of 124 subjects were treated with DARZALEX ^™ (dalazumab) (de Weers et al., (2011) J Immunol 186(3): 1840-1848). 18 subjects were treated at 8 mg/kg, and 106 subjects were treated at 16 mg/kg. The dosing schedule is as follows: Group A: DARZALEX ^TM (dalazumab) 16 mg/kg: Cycle 1 and Cycle 2: Days 1, 8, 15, and 22 (weekly), Cycle 3 to Cycle 6: Day 1 and 15 days (every other week), cycle 7+: Day 1 (every 4 weeks). Each cycle is 4 weeks.

Group B: DARZALEX ^TM (Dalamumab) 8 mg/kg: Cycle 1+: Day 1 (every 4 weeks).

The main goal of the study is to determine ^{the efficacy of the two treatment regimens of DARZALEX TM} (dalazumab). As measured by ORR (CR+PR), in subjects with multiple myeloma, these subjects have previously Receiving at least 3 therapies, including PI and IMiD, or the diseases of these subjects are double refractory to PI and IMiD (clinical research report: an open-label, multi-center, phase 2 trial, which explores the ^{Efficacy and safety of DARZALEX TM} (dalazumab) in subjects with myeloma, who have previously received at least 3 therapies (including proteasome inhibitors and IMiD) In terms of double refractory. EDMS-ERI-92399922).

The secondary goals of this study include assessing ^{the safety and tolerability of DARZALEX TM} (dalazumab), demonstrating additional measures of efficacy (for example, clinical benefit, TTP, PFS, and OS), as well as assessing pharmacokinetics, immunogen Properties, pharmacodynamics, and exploration of biomarkers predicting response to DARZALEX ^TM (dalazumab). Additional research-related information can be obtained from the clinical research protocol (clinical research report: an open-label, multi-center, phase 2 trial that explores the effects of DARZALEX ^TM (dalazumab) in subjects with multiple myeloma) Efficacy and safety, these subjects have previously received at least 3 therapies (including proteasome inhibitors and IMiD) or these subjects are double refractory to proteasome inhibitors and IMiD. EDMS-ERI-92399922).

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

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

Example 3. DARZALEX ^TM (Dalazumab) Effect on T cell expansion and activity in patients enrolled in the 54767414MMY2002 study (SIRIUS)

CD38 is expressed on a variety of immune cells and hematopoietic cells. Extensive immunoassays were performed by flow cytometry to test the effect of DARZALEX ^TM (dalazumab) on a subset of immune cells and the correlation between the baseline level of these cells and clinical response. After the base phase and DARZALEX ^TM (dalazumab) treatment, flow cytometry is used to evaluate various cell populations in peripheral blood and bone marrow aspirates, including T cells (CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , and regulatory T cells) (Treg)), B cells (CD19 ⁺ ), NK cells, monocytes (CD14 ⁺ ), white blood cells, and neutrophils to monitor changes in these cell populations in responders and non-responders.

Lymphocytes, white blood cells, monocytes, and neutrophils

Study the counts of leukocytes, lymphocytes, monocytes, and neutrophils in peripheral blood in responders and non-responders. In patients at doses of 8 mg/kg and 16 mg/kg, total lymphocytes were found to increase with DARZALEX ^TM (dalazumab) treatment (Figure 1) . Linear mixed effect modeling revealed an increase of 0.8×10 ⁶ cells/μL on a logarithmic scale every 100 days (CI=0.06, 0.11). It was found that monocytes and white blood cells increased slightly, which were significantly increased by 0.03×10 ⁶ cells/μL (CI=0.01, 0.04) and 0.03×10 ^{6 cells/μL (CI=} 0.01, 0.05). Although it was noted that neutrophils decreased in some patients, the median neutrophil count was consistent with the base period, and the change was not significant.

Compare the baseline level of each of these cell populations between the response groups. Using the Wilcoxon signed-rank test did not find evidence that the baseline levels of any of these cell types across the response group were different ( Table 1 ).

NK cells

Total NK cells (CD16 ⁺ CD56 ⁺ ) and activated NK cells (CD16 ⁺ CD56 ^dim ) decreased over time under DARZALEX™ (data not shown).

B cell

The absolute counts ^{of B cells (CD45 +} CD3 ^- CD19 ⁺ ) in ^{peripheral blood or bone marrow aspirates during DARZALEX TM} (dalazumab) treatment were measured in responders and non-responders over time. B cells increased slightly in whole blood and remained unchanged in bone marrow aspirates. The linear mixed modelling of B cells in the peripheral blood revealed that the ^{minimum increase of 0.1×10 6} cells/μL on the logarithmic scale during each 100 days of ^{DARZALEX TM (dalazumab) treatment process [CI=0.04,0.16} ]. During dalamumab treatment, the percentage of B cells (CD45 ⁺ CD3 ^- CD19 ⁺ /lymphocytes) in bone marrow aspirates did not change among responders or non-responders (p=0.1 and 0.4, respectively). In addition, between responders and non-responders, there was no evidence of differences in B cell counts in the base phase (p=0.5).

T cells noticed the increase in lymphocytes under DARZALEX ^(TM ) treatment ( Figure 1 ), even though B cells showed only minimal increase (see above). For further investigation, various T cell populations (CD3 ⁺ , CD4 ⁺ , CD8 ⁺ T cells, regulatory T cells) in both peripheral blood and bone marrow were studied.

After DARZALEX ^TM (dalazumab) treatment, CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ T cells in the peripheral blood increase (both absolute count of lymphocytes/μl and percentage). Figure 2 shows the percentage change of the absolute count ^{of CD3 +} T cells (CD45 ⁺ CD3 ⁺ ) in the peripheral blood of each patient from the baseline over time. The black line in the figure shows the median absolute count of all patients×10 ⁶ cells/μL. The figure only includes visits with more than 2 observations. Figure 3 shows the% change in the absolute count ^{of CD4 +} T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ ) in the peripheral blood of each patient over time from the baseline. The black line in the figure shows the median of all patients. The figure only includes visits with more than 2 observations. Figure 4 shows the% change in the absolute count ^{of CD8 +} T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ ) in the peripheral blood of each patient over time from the baseline. The black line in the figure shows the median of all patients. The figure only includes visits with more than 2 observations. Linear mixed modeling of absolute counts in peripheral blood reveals that, on average, total T cells (CD45 ⁺ CD3 ⁺ ) after DARZALEX ^TM (dalazumab) treatment increased by 0.13× on a logarithmic scale every 100 days. 10 ⁶ cells/μl (CI=0.1,0.15). It was found that CD8 ⁺ T cells increased significantly by 0.16×10 ⁶ cells/μl (CI=0.13, 0.19) on a logarithmic scale on each 100 days. It was found that CD4 ^{+ cells increased by 0.11×10 6} cells/μl on a logarithmic scale every 100 days (CI=0.09, 0.13).

For each of the T cell subpopulations, the responders showed that the maximum percentage change in absolute counts from the base period was higher than that of the non-responders (CD3 ⁺ p=3.2993e-05; CD4 ⁺ p=3.486e-05; CD8 ⁺ p=2.7172e -05; regulatory T cells p=0.002). Table 2 shows the results of Wilkason's sign rank test to compare the percentage change from the baseline of the absolute count of each T cell subpopulation in the peripheral blood between responders and non-responders.

Similarly, in the bone marrow, the total T cells (CD45 ⁺ CD3 ^{+ as} the percentage of lymphocytes) and CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ^{+ as} the percentage of lymphocytes) of responders and non-responders are found in DARZALEX ^TM (dalazumab) increased significantly during treatment (CD3 ⁺ responders p=3.8147e-06, non-responders p=9.8225e-05; CD8 ⁺ responders p=3.8147e-06, non-responders p =0.0003). The median number of CD4 ⁺ T cells in the bone marrow did not change in any clinical response group. Table 3 shows the results of Wilcarson's sign rank test of various T cells showing% of lymphocytes in bone marrow. Figure 5 shows ^{the percentage (%) of CD45 +} CD3 ^{+ cells over time during DARZALEX TM} (Dalamumab) treatment (both responders and non-responders are included in the chart). Figure 6 shows the% of CD45 ⁺ CD3 ⁺ CD8 ^{+ cells over time during DARZALEX TM} (Dalamumab) treatment (both responders and non-responders are included in the chart).

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

According to Wilcarson's symbolic grade test, the absolute T cell count in the peripheral blood at the base period ( Table 4 ) or the percentage of T cells from the total lymphocytes in the bone marrow ( Table 5 ), between responders and non-responders There is no statistically significant difference.

T regulatory cells

The Treg cell line was identified as the CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim cell population in the sample. To evaluate ^{the ratio of CD8 +} T cells to Tregs in peripheral blood and bone marrow over time in patients treated ^{with DARZALEX TM (dalazumab).} The ratio in peripheral blood and bone marrow increased. Figure 7A ^{shows the median values of the CD8 +} /Treg and CD8 ^+/ CD4 ⁺ cell ratios in the peripheral blood of all patients at each time point. Figure 7B ^{shows the median CD8 +} /Treg and CD8 ⁺ /CD4 ⁺ T cell ratios in the bone marrow of all patients at each time point. According to Wilcarson's symbolic grade test, the changes in the absolute count ratios of ^{CD8 +} Treg and CD8 ⁺ /CD4 ⁺ in peripheral blood with the passage of treatment time ( Table 6 ) and bone marrow ( Table 7 ) are significant.

In the combined data analysis of the SIRIUS and GEN501 study (Example 6) ^{, the median ratio of CD8 +} /CD4 ⁺ and CD8 ⁺ /Treg cells in peripheral blood was at week 8 (CD8 ⁺ /CD4 ⁺ p=5.1× 10 ^-5 and CD8 ⁺ /Treg p=1.8×10 ^-7 ) and increase in week 16 (CD8 ⁺ /CD4 ⁺ p=0.00017 and CD8 ⁺ /Treg p=4.1×10 ^-7 ) of. Similarly, in the bone marrow, the ^{median ratio of CD8 +} /CD4 ⁺ and CD8 ⁺ /Treg cells increased during treatment (the 12th + 1 week cycle) compared to the baseline (CD8 ⁺ /CD4 ⁺ p= 0.00016, and CD8 ⁺ /Treg's p=2.8×10 ^-7 ). No significant difference was observed between responders and non-responders.

Example 4. Research Design (GEN501)

^{Study GEN501 (NCT00572488) evaluates DARZALEX TM} (dalazumab) as a monotherapy in patients with dual refractory MM. Sample isolation, processing, and statistical analysis are as described in Example 1 and Example 2. This study has been described in Lokhorst et al., N Eng J Med 373:1207-19, 2005.

In short, the study of GEN501 is the first-in-human clinical study of DARZALEXTM (darazumab) in subjects suffering from MM. It is the Phase 1/2 safety study of escalating doses. The study is divided into two parts. Part 1 is a study on the gradual increase in open-label doses; Part 2 is a single-arm study of open-label groups in multiple groups, based on the dose levels established in Part 1.

In Part 1, ^{10 dose levels of DARZALEX(TM)} (dalazumab) are evaluated: 0.005, 0.05, 0.10, 0.50, 1, 2, 4, 8, 16, and 24 mg/kg. The two lowest dose groups were each allocated to 1 (+3) subjects, and the standard 3 (+3) subject ration was applied to the remaining 8 dose groups. Part 2 is an open-label single study, which includes two dose levels, 8mg/kg and 16mg/kg. Part 1 includes 32 objects, and Part 2 includes 72 objects.

Example 5. DARZALEX ^TM (Dalazumab) treatment induces homologous T cells in patients

PR；無反應者n=11，即，MR、SD、PD)。 ^{In view of the expansion of CD8 +} T cells noted in the peripheral blood and bone marrow in the MY2002 study, the Immunoseq ^TM assay was used to perform high-throughput next-generation sequencing of T cell receptors (TCR) to determine the expansion of CD8 ⁺ T Whether a cell is pure in nature is an indicator of an adaptive immune response. Evaluate a total of 17 patient samples from subjects registered in the GEN501 study (respondents n=6, that is,

PR; non-responders n=11, that is, MR, SD, PD).

TCR sequencing revealed that DARZALEX ^(TM ) treatment significantly increased the homology of the patient. Figure 8A shows ^{the correlation between T cell homotypic before and after DARZALEX™} (dalumab) treatment (p=0.0056). Figure 8B shows the fold change of homology in individual patients. Respondents are marked with stars. This data indicates that the T cell expansion noted in the context of ^{DARZALEX(TM) treatment can be pure-line in nature.}

When compared to non-responders, responders have greater total amplification in the TCR reservoir (as measured by changes in abundance; CIA). Figure 8C shows the CIA% of individual patients. Group A: responders, group B: non-responders. A statistically significant difference was observed between responders and non-responders (p=0.037). Figure 8D shows the sum of the absolute abundance change (CIA) of each expanded T cell clone in responders and non-responders. Figure 8E shows the maximum CIA% for each individual patient. Group A: responders, group B: non-responders. A statistically significant difference was observed between responders and non-responders (p=0.048). Figure 8F shows the maximum CIA of single cell clones in responders (group A) and non-responders (group B).

The CIA line uses Fisher's exact test (DeWit et al. J.Virol. 2015) to identify significant differences in the abundance of pure lines between the two samples and changes in the absolute abundance of each expanded strain Sum to get.

Example 6. DARZALEX ^TM (Dalazumab) Immunomodulatory effect in patients enrolled in the GEN501 study

Assess various T and B cell populations in responders and non-responders registered with GEN501.

Lymphocytes

Similar to the SIRIUS (MMY2002) study, during the DARZALEX ^TM (dalazumab) treatment, lymphocytes in the peripheral blood and bone marrow increased. This increase is due to the increase in the number of both ^CD4+ and CD8 ^{+ cells.}

CD8 ⁺ Central memory cell

A subset of 17 patients who registered for the GEN 501 study focused on ^{the CD8 +} T cell phenotype over time in patients treated ^{with DARZALEX TM (dalazumab).} Using standard procedures, CD8 ⁺ cells from the patient were identified as naive (CD45RO-/CD62L ⁺ ) (T _N ) cells or central memory (T _CM ) (CD45RO ⁺ /CD62L ^{+ high} ) cells.

MR)之病患，且黑色方形指示疾病穩定或疾病進展之病患。對治療有反應的病患中，CD8⁺初始T細胞之顯著地較大的降低係明顯的(數據未顯示)。圖9C顯示，DARZALEX^TM(達拉單抗)治療增加HLA I類-限制性T細胞之百分比，該等細胞部分驅動病毒特異性及同種反應性T細胞反應。圖9D顯示，擴增效應記憶T細胞表現低水平的CD38。重要的是要注意，這些T細胞呈現正常且甚至增加的針對病毒胜肽及同種抗原的功能活性(參見實例8)。根據這些功能結果，我們推斷，在DARZALEX^TM(達拉單抗)治療期間存在針對病毒及同種抗原的經歷抗原之T細胞的擴增、或活性改善。這些數據表明，不同於調節細胞子集，效應T細胞不需要CD38表現以適當地起作用並擴增。 Figure 9A shows ^{the% of CD8 +} naive cells (% of CD8 ⁺ cells), and Figure 9B shows ^{the% of CD8 +} central memory cells. DARZALEX ^TM treatment significantly reduced the amount of ^{initial CD8 +} T cells (p = 1.82×10 ^{-4 at} the 8th week) and increased the amount of CD8 ⁺ memory T cells ( p = 4.88 at the 8th week) ×10 ^-2 ). This indicates that naive cytotoxic T cells are transformed into memory T cells, which can be activated against specific antigens. The white square indicates that at least the minimum response has been achieved (

MR) patients, and the black squares indicate patients with stable disease or disease progression. In patients who responded to treatment, a ^{significantly greater reduction in CD8+} naive T cells was evident (data not shown). Figure 9C shows that DARZALEX ^(TM ) treatment increases the percentage of HLA class I-restricted T cells, which partially drive virus-specific and alloreactive T cell responses. Figure 9D shows that expanded effector memory T cells exhibit low levels of CD38. It is important to note that these T cells exhibit normal and even increased functional activity against viral peptides and homologous antigens (see Example 8 ). Based on these functional results, we infer that there is an expansion of antigen-experienced T cells against the virus and alloantigens, or an improvement in activity during ^{DARZALEX TM (dalazumab) treatment.} These data indicate that, unlike a subset of regulatory cells, effector T cells do not require CD38 expression in order to function and expand properly.

CD38 positive regulatory T cells

The observation of the robust expansion and increased activity of cytotoxic T cells, together with recent literature indicating that a subset of immunosuppressive cells express CD38, facilitates the examination of DARZALEX ^TM (dalazumab) on regulatory cell populations regulatory T cells (Treg) , Bone marrow-derived suppressor cells (MDSC), and regulate the effects of B cells (Breg).

Isolate regulatory T cells (Treg) using standard procedures (CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ). Use flow cytometry to analyze the frequency of Treg.

Before Treg activation, a subset of peripheral Tregs (10%+10%) showed high levels of CD38. Figure 10A, the upper panel shows the frequency of Treg in the CD3 ⁺ CD4 ⁺ cell population (P4 cell population) at the base phase. Figure 10A , the bottom panel shows a subset of Tregs (P5 cell population) that exhibit high CD38. These CD38 ⁺ Tregs are ^{highly sensitive to DARZALEX TM} (dalazumab) treatment, and showed significant and almost immediate decline after the first dose of ^{DARZALEX TM (dalazumab) (patient n=17; 1st} Weekly to the base period P = 8.88×10 ^-16 ). The frequency of Treg after DARZALEX ^(TM ) treatment is shown in Figure 10B , in the upper panel (P4 cell population). Figure 10B, the bottom panel shows the Treg population with the most significant loss of the ^{CD38 high} Treg (P5 cell) line after the ^{first (1 st} ) DARZALEX ^{™ (dalumab) infusion.} These CD38 ⁺ Tregs were maintained attrition during the entire DARZALEX ^TM (dalazumab) treatment (p =8.88×10 ^-16 , 1.11×10 ^-15 , and p = 8.88×10 -16, 1.11×10 -15, and at the base period at the first, 4, and 8 weeks, respectively 1.50×10 ^-11 ). Figure 10C shows the% of CD38 ^{high Treg of total CD3+} cells 6 months after base period, week 1, week 4, week 8, relapse, and end of treatment (EOT). CD38 ^high Treg recovered to the base period at this point in time. ^{The changes in CD38 +} Treg between patients who responded to treatment and those who did not respond ^{were similar. However, the CD8 +} T cells of patients who showed a response to DARZALEX ^TM (dalazumab) treatment at week 8: The Treg ratio was significantly higher ( P =0.00955; Figure 10D ).

In order to evaluate the possible biological correlation between the loss of ^{CD38 + Treg and DARZALEX TM (darumab) treatment, the ability of CD38 +} Treg ^{on autologous CD3 +} T cells to ^{inhibit CD38-} Treg was evaluated. In a series of experiments using samples from multiple healthy donors, CD38 ⁺ Treg inhibited T cell proliferation (9.9% cell proliferation was observed) than CD38 ^- Treg (53.2% cell proliferation was observed) or a negative control (observed To 74.9% cell proliferation) is more robust ( Figure 10E ).

Because MDSC is not easy to detect in frozen PBMC samples, CD38 ⁺ granular spherical MDSC (CD11b ⁺ CD14 ^- HLA-DR ^- CD15 ⁺ CD33 ⁺ ) is generated from PBMC in vitro. Patients who received a DARZALEX ^TM (dalazumab) infusion were isolated. Figure 11 shows the flow cytometry distribution map of the identified MDSCs ( Figure 11 , upper distribution map, boxed cell population). Approximately half of MDSCs express CD38 ( Figure 11 , middle graph; circled P7 cell population). In patients treated with DARZALEX ^TM (dalazumab), the CD38 ^high MDSC line was almost depleted ( Figure 11 , bottom graph; circled P7 cell population).

Among non-responders and patients with at least minimal repose to treatment, CD38 ^high- spectrum non-specific MDSCs are depleted over time under ^{DARZALEX TM (dalazumab) treatment.} Figure 12 shows that at 1 week, 4 weeks, or 8 weeks of treatment, ^{the percentage of CD38 high} MDSC in the patients decreased to nearly 0%. CD38 ^high- lineage non-specific MDSC returned to the base stage after the treatment.

^{Patients with the largest CD38+} population within the lineage non-specific MDSC exhibited the best and most durable response to DARZALEX ^{(TM) treatment.} Figure 13 shows that patients 2, 4, 15, 16, or 17 with the highest percentage of CD38 ^high MDSC (as shown in Figure 11) and classified as patients with PR or MR have at least 8 months of progression-free Survival period (Progression-Free Survival, PFS).

CD38 ^high- lineage non-specific MDSCs are also sensitive to ADCC induced by ^{DARZALEX TM} in vitro. ^{CD38 high} MDSC and Daudi cells from two donors were used as control target cells for ADCC determination, in which the ratio of effector cells: target cells was 50:1. Figure 14 shows the results of an experiment from a donor. DARZALEX ^(TM) (dalazumab) induces the lysis of MDSC cells.

^{CD38 +} Breg was measured in patients (n=16) treated with DARZALEX ^TM (dalazumab), and similar to CD38 ⁺ Treg, CD38 ⁺ Breg was depleted after the first dose of ^{DARZALEX TM (dalazumab)} (Compared with base period in week 1, p = 0.0018; paired Wilkason rating test) and continued to be low when the patient was on treatment ( Figure 15A ). When stimulated, Breg sorted by FACS produces IL-10 ( Figure 15B ).

In summary, these observations indicate that ^{the depletion of immunosuppressive CD38 +} MDSCs, Bregs, and Tregs is a significant contributory mechanism for the changes in T cell populations and ^{clones induced by DARZALEX(TM).}

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

^{Use flow cytometry to study the percentage of MDSC (Lin-} CD14 ^- HLADR ^low/- ) and other CD38 manifestations in the peripheral blood of patients with NSCLC or prostate cancer.

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

Example 8. DARZALEX ^TM (Dalazumab) enhances antiviral T cell response

In order to further evaluate the effect of DARZALEX ^TM (dalazumab) on T cell activation and function, in ^{patients (n=7) treated with DARZALEX TM} (dalazumab) with a certain range of clinical outcomes, the measurement response was IFN-γ production by surrounding T cells in viruses and allogeneic antigens. Patients with PR or better response after DARZALEX ^TM (dalazumab) treatment showed a significant increase in IFN-γ secretion in response to the virus and alloantigens compared to the base phase, at least at one time point during the treatment period This is so, which indicates that T cell function is not impaired by low CD38 manifestations (see Example 6, Figure 9C). Similar to the TCR pure lineage data, this increase ^{was more pronounced in patients who responded to DARZALEX TM} (dalazumab) than in patients who did not respond. Figure 16A shows the antiviral response of a representative patient with VGPR. Figure 16B shows the antiviral response of a representative patient with CR. Figure 16C shows the antiviral response of a representative patient with PD. Figure 16D shows the antiviral response of a representative patient with MR. In the diagram, the error bars represent the standard error of the average of the duplicate cultures. Asterisks indicate statistically significant changes between the indicated comparisons. Shows the best response in accordance with the standards of the independent review committee. Consistent with these results, virus-reactive T cells in patients with VGPR (Figure 16E) or CR (Figure 16F) showed an increase in proliferative capacity during ^{DARZALEX(TM) treatment.}

Example 9. Immune cell subtypes expressing CD38 vs. DARZALEX ^TM (Dalazumab) sensitivity mechanism

The data from the GEN501 and SIRIUS studies indicate that under DARZALEX ^TM (dalazumab) therapy, there are some immune cell lines that exhibit CD38 depletion (NK cells, regulatory T cells (Treg), regulatory B cells (Breg), and bone marrow). Derived suppressor cells (MDSC)), however, the number of other immune cells that express CD38 is increased (cytotoxic T cells and helper T cells).

In order to solve the mechanism of sensitivity, the expression level of CD38 in various immune cell subpopulations in healthy donors and in patients with multiple myeloma registered in the GEN501 or SIRIUS study was evaluated. Figure 17A shows the distribution of CD38 expression in immune cells of healthy donors, and Figure 17B shows the distribution of CD38 expression in immune cells of patients with multiple myeloma. Among healthy donors, CD38 was highest on NK cells, followed by monocytes, B cells, and T cells. In patients with multiple myeloma, CD38 is highest on plasma cells, followed by a subset of B cells, NK cells, monocytes, B cells, and T cells. Figure 17C shows the comparison of the CD38 mean fluorescence intensity (MFI) of NK cells, Treg, Breg, B cells, and T cells in patients with relapsed and refractory myeloma. It shows that after plasma cells, NK cells have the highest performance The level of CD38 is followed by regulatory T cells (Treg) and regulatory B cells (Breg).

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

discuss

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

This study demonstrates that MDSC and Breg express CD38 and are susceptible to DARZALEX ^TM (dalazumab) treatment. These cells are known to exist in the tumor microenvironment and cause tumor growth, immune evasion, angiogenesis, metastasis, and the production of inhibitory cytokines. In addition to these CD38 ⁺ inhibitory cell subsets, a novel regulatory T cell subset (CD4 ⁺ CD25 ⁺ CD127 ^dim ) has also been identified, which also exhibits high levels of CD38 and exhibits excellent autologous T cell inhibitory ability. These cells are also ^{sensitive to DARZALEX(TM)} (darumab), and in patients receiving treatment, these cells are significantly reduced. The elimination of these CD38 ⁺ immunoregulatory cells mediated by DARZALEX ^TM (dalazumab) can reduce local immunosuppression in the myeloma microenvironment, and allow positive immune effector cells to expand and lead to anti-tumor responses.

In fact, a significant increase in a broad T cell population ^{including CD4 +} and CD8 ^{+ was} observed in the peripheral blood and in the bone marrow (i.e., tumor). Specific CD8 ⁺ subpopulations have been altered by DARZALEX ^TM (dalazumab) therapy, including a significant reduction in naive T cells and a concomitant significant increase in effector memory CD8 ⁺ T cells, which indicate a shift of effector T cells to a phenotype experiencing antigen This phenotype retains immune memory and can be reactive to tumor antigens. The ratio of CD8 ⁺ :CD4 ⁺ and CD8 ⁺ : Treg also increased significantly under treatment, which showed a shift of positive immunomodulator from negative immunomodulator.

In order to assess whether the expanded CD4 ⁺ and CD8 ⁺ T cells are pure in nature, examine the T cell reservoir in a subset of patients. Even in patients with the best response to SD or patients with progression, T cell homology is significantly increased under ^{DARZALEX TM (dalazumab) treatment.} Therefore, the increase in homologous T cells cannot be simply attributed to the decrease in tumor burden. However, the deviation of T cell homology is greater in patients with good clinical response and is associated with ^{an increase in CD8 +} T cells, which indicates the expansion of T cells observed under ^{DARZALEX TM (dalazumab) treatment.} Proliferation of antigen-driven. This is significant in this patient population, which has undergone a large number of pre-treatments (median is 5 previous therapies) and is not expected to be able to establish a strong anti-tumor immune response. In addition to increased TCR homology ^{, patients who are responsive to DARZALEX TM} (dalazumab) exhibit increased T cell responses to pre-existing viruses and alloantigens, which indicates the rescue of the immune system from an immunosuppressive state.

The use of DARZALEX ^TM (Dalazumab) treatment resulted in a reduction of immunosuppressive MDSC and regulatory T cells and B cells. These reductions are accompanied by ^{the expansion of CD4 +} T helper cells and CD8 ⁺ cytotoxic T cells. The T cell homologous and functional antiviral response as measured by IFN-γ production was also increased under DARZALEX ^TM (dalazumab) treatment. These observations indicate that T cells continue to function properly despite low CD38 performance, and indicate that the increased T cell response may be due to the depletion of regulatory cells. In addition, these T cell expansion, activity, and homologous changes may be more significant in patients who responded ^{to DARZALEX TM (dalazumab) than in patients who did not respond.} Recurrence from DARZALEX ^TM (dalazumab) therapy is associated with the reversal of many of these changes. This indicates ^{the effect of the additional, previously uncharacterized mechanism of DARZALEX TM} (dalazumab) through immunomodulation, which can lead to clinical response and the efficacy of DARZALEX ^TM (dalazumab).

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

Example 10. In 54767414MMY2002 (SIRIUS) Part 2 clinical study with single-dose DARZALEX ^TM (Dalazumab) Serum proteosome analysis of interchangeable multiple myeloma subjects Biomarker sample collection and processing

The peripheral blood samples were collected in standard serum separation tubes (2.5 mL to 5 mL), and the serum samples were aliquoted frozen and transported to SomaLogic, Inc (Boulder, CO) for multi-analyte serum protein analysis.

Serum protein profiling is performed at SomaLogic using the pre-validated SOMAscan assay, which measures 1129 protein analytes using SOMAmer affinity-based molecules. SOMAmer reagent is a protein affinity reagent based on single-stranded DNA. The assay uses a small amount of input sample (150 μL of plasma) and converts the protein signal into a SOMAmer signal, which is quantified by a customized DNA microarray.

Each SOMAmer contains 4 functional parts:

1. A unique protein recognition sequence

2. Biotin for capture

3. Photocleavable linker

4. Fluorescent molecules for detection

The unique protein recognition sequence uses DNA and incorporates chemically modified nucleotides that mimic amino acid side chains, which amplify the diversity of standard aptamers and enhance the specificity and affinity of protein-nucleic acid interactions (Gold et al., PLoS One 5: e15004, 2010). The suitable system is selected by SELEX. The SOMAmer reagent is selected using the protein in the original configuration. Because this type of SOMAmer reagent requires a complete tertiary protein structure to bind. Unfolded or denatured presumably inactive proteins are not detected by SOMAmer reagent.

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

The samples from the MMY2002 study were tested in two main batches. The 180 samples in the first batch contained a pair of serum samples from 90 subjects on Day 1 (C1D1, Base Period) and C3D1 (Day 1 of Cycle 3) from 90 subjects. The 180 samples were analyzed together on 3 separate SomaScan discs. The second batch of samples included 50 C1D1 samples, which included 35 replicate samples from the first batch.

data analysis Input data set and definition

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

Somalogic data preprocessing Batch calibration

The first and second batch of MMY2002 samples were tested on two different versions of the SOMAscan platform. The differences between the two versions are small and include three SOMAmer sequences, which have changed between versions (CTSE: 3594-6_1->3594-6_5, FCN1: 3613-62_1->3613-62_5, BMPER: 3654-27_1->3654-27_4). These are removed from the analysis.

According to SomaLogic's standard inter-plate calibration workflow, the plate-wide of each SOMAmer is defined by calculating the ratio of the specific overall reference value of the master mix (Master-mix) to the measured values of the 7 intra-plate control calibrators. The scale factor is calibrated, so the measured values of the three first batch disks are calibrated. The plate-specific scale factor of each SOMAmer reagent is equally applied to each sample on the plate.

。然後將這些校準比例因數同樣地實施於標準SOMAscan程序。 Given the different SOMAscan platform versions of the first batch and the second batch, by considering the ratio of the repeated measurement values of 35 samples across batches, using the modified implementation scheme of SomaLogic's inter-standard calibration workflow Systematic correction for batch-to-batch variability. For each SOMAmer, calculate the ratio (ri,j ) of the measured value after the first batch of calibration divided by the measured value of the second batch of each of the 35 replicate samples. Use the median of these 35 ratios to define the modified SOMAmer specific calibration ratio factor for the second batch of samples

. Then these calibration scale factors are also implemented in the standard SOMAscan program.

Once the corrected calibration scale factor is calculated, the distribution of all scale factors for each batch of analysis is drawn to assess the existence of outliers. Due to poor reproducibility, 9 SOMAmers with very large or very small calibration values (>0.25 and <3) were removed from the analysis.

After batch calibration of MMY2002 and SOMAmer filtering are completed, log2 conversion is applied to all protein concentration values of MMY2002 to make the data more in line with the normal distribution and improve the performance of parameter statistical verification.

Confounding Variable Correction

7.37%，p-值=3.71×10-9)。為了減少樣本獲得部位相關效應在數據內的影響，利用ComBat28校正部位ID效應。 By principal component analysis of a centered and proportional data set, estimate the part of the data set variance explained by meta-variables (such as demographics, response levels, and sample time points) And the identification of possible confounding factors. A simple linear model is fitted to identify the highest grade PC that is significantly associated with each variable of interest. The significance of these associations was determined using the Wald test, and the part of the PC variability explained by the model was estimated by the fitted R2. For the MMY2002 data, the part ID was found to be related to PC1 and explained the largest part of the variability of the data set (

7.37%, p-value = 3.71×10-9). In order to reduce the influence of the position-related effects of sample acquisition in the data, ComBat28 is used to correct the position ID effect.

Duplicate sample merging

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

Differential Protein Concentration Analysis (Differential Protein Concentration Analysis) responders vs. non-responders

The statistical comparison of the protein concentration distribution between DARZALEXTM responders and non-responders in the base phase and during treatment was carried out using two complementary methods: (i) Wilkason rank and test ( Hollander and Wolfe, Ninparametirc Statistical Methods. New York: John Wiley & Sons. 1973.27-33 (one sample), 68-75 (two samples), performed on each SOMAmer, and (ii) Limma analysis (Ritchie, ME, etc.) Human, Nucleic Acids Res. 2015; 20: 43(7): e47), performed simultaneously for all SOMAmers. All p-value systems use Benjamini-Hochberg (Benjamini-Hochberg; BH) for multi-hypothesis correction Method to adjust (Benjamini and Hochberg, (1995) JRStatist.Soc. B.57: 289-300; R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria. 2011 ; ISBN 3-900051-07-0). When the adjusted p-value is <0.05, the zero hypothesis of no difference in performance is rejected.

Treatment in the base period

Three alternative statistical methods were used to compare the baseline protein level to the protein level in treatment: (i) two-way repeated measures ANOVA6, (ii) Wilkason sign rank test, and (iii) Friedman test (Friedman test) ( Johnson et al. (2007) Biostatistics 8(1):118-127). All p-values were adjusted to control FDR, and the adjustment was performed using the BH method for multi-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) is also used to determine each SOMAmer Does a significant point in time occur: reaction level interaction. The modified Wilkason level and test are used as post-mortem tests to clearly determine whether responders and non-responders show different therapeutic effects by calculating the difference between the protein concentration value during treatment and the base-phase protein concentration value for each subject The difference and perform Wil Carson's level and verification. Use the BH method to adjust the significance value, and when the adjusted p-value is <0.05, the zero hypothesis is rejected.

Grading system training

The baseline protein level MMY2002 data was used to establish a response prediction grading system. The 10-fold cross-validation approach of nesting loop layering is repeated 30 times, using 4 different machine learners: Support Vector Machine (SVM), Random Forest (Random Forest) ,RF), Naïve Baye (NB), and j48 decision tree. For each learner, the training program starts with 10 folds that generate the balance of the data set (outer loop). One of these folds is provided as a test cluster, and the remaining 9 are passed to the inner loop as a training cluster. In the inner loop, the training group is again divided into 10 balanced folds, which generate the training inner set and the test inner set. Right this Each of these training inner sets performs learner training, and this procedure is repeated 30 times for each group in the outer loop. The accuracy of each inner loop learner in predicting the inner set of the test is used to select features and optimize model parameters. Once the 30 times (30x) inner loops of each training group are completed, the performance of the outer loop (using optimized parameters and features) is evaluated for each corresponding test group. Then the entire outer loop procedure was repeated 30 times, and 30 reaction predictions were generated for each sample in the data set. The AUC, sensitivity, and specificity statistics obtained from this round-robin method are the approximate values of the performance of the final model (trained with the complete original data set) on the new test case.

MMY2002 research results

Various comparisons were made, including treatment-induced response-dependent changes in protein expression. Among the responders, one of the proteins that showed decreased performance over time was PD-L1; however, among non-responders, the protein performance of PD-L1 increased over time. The engagement of PD-L1 on T cells causes a decrease in T cell function and an increase in Treg development. Figure 18 shows the profile of PD-L1 protein expression in responders, non-responders, and patients with stable disease in cycles 1 and 3.

The engagement of PD-L1 with its receptor PD-1 inhibits the anti-tumor response and drives T cell anergy and depletion. Although not wishing to be bound by any specific theory, after CD38 treatment, PD-L1 down-regulation may also lead to an improvement in the anti-tumor immune response of solid tumors.

<110> Janssen Biotech, Inc.

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

<140> 105134914

<141> 2016-10-28

<150> US 15/191808

<151> 2016-06-24

<150> PCT/US 16/39165

<151> 2016-06-24

<150> US 62/331489

<151> 2016-05-04

<150> US 62/263307

<151> 2015-12-04

<150> US 62/250566

<151> 2015-11-04

<150> US 62/249546

<151> 2015-11-02

<150> US 62/263199

<151> 2015-12-04

<160> 40

<170> PatentIn version 3.5

<210> 1

<211> 300

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 14

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 14

<212> PRT

<213> Homo sapiens

<400> 3

<210> 4

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-CD38 antibody

<400> 4

<210> 5

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of anti-CD38 antibody

<400> 5

<210> 6

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR1 of anti-CD38 antibody

<400> 6

<210> 7

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR2 of anti-CD38 antibody

<400> 7

<210> 8

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> HCDR3 of anti-CD38 antibody

<400> 8

<210> 9

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR1 of anti-CD38 antibody

<400> 9

<210> 10

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR2 of anti-CD38 antibody

<400> 10

<210> 11

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> LCDR3 of anti-CD38 antibody

<400> 11

<210> 12

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain of anti-CD38 antibody

<400> 12

<210> 13

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain of anti-CD38 antibody

<400> 13

<210> 14

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH 003 of anti-CD38 antibody

<400> 14

<210> 15

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL 003 of anti-CD38 antibody

<400> 15

<210> 16

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH 024 of anti-CD38 antibody

<400> 16

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL 024 of anti-CD38 antibody

<400> 17

<210> 18

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH MOR202 of anti-CD38 antibody

<400> 18

<210> 19

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> VL MOR202 of anti-CD38 antibody

<400> 19

<210> 20

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH Isartuximab with anti-CD38 mAb

<400> 20

<210> 21

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL Isartuximab against CD38 mAb

<400> 21

<210> 22

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> VH Keytruda of anti-PD-1 mAb

<400> 22

<210> 23

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> VL Keytruda of anti-PD-1 mAb

<400> 23

<210> 24

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> VH Opdivo of anti-PD-1 mAb

<400> 24

<210> 25

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL Opdivo of anti-PD-1 mAb

<400> 25

<210> 26

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH Devalumab against PD-L1 mAb

<400> 26

<210> 27

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL Devalumab for anti-PD-L1 mAb

<400> 27

<210> 28

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> VH atezizumab against PD-L1 mAb

<400> 28

<210> 29

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL atezizumab for anti-PD-L1 mAb

<400> 29

<210> 30

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 mAb of VH Avilizumab

<400> 30

<210> 31

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-PD-L1 mAb VL Avillumab

<400> 31

<210> 32

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-PD-1 mAb

<400> 32

<210> 33

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of anti-PD-1 mAb

<400> 33

<210> 34

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-PD-1 mAb

<400> 34

<210> 35

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-PD-1 mAb

<400> 35

<210> 36

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-TIM-3 mAb

<400> 36

<210> 37

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VL of anti-TIM-3 mAb

<400> 37

<210> 38

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> VH of anti-TIM-3 mAb

<400> 38

<210> 39

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VL of anti-TIM-3 mAb

<400> 39

<210> 40

<211> 509

<212> PRT

<213> Artificial sequence

<220>

<223> Recombinant Hyaluronidase

<400> 40

Claims (59)

The use of an antibody that specifically binds CD38 in the manufacture of a drug for the treatment of solid tumors in patients, wherein the antibody comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and SEQ ID NO: 6, 7 and 8, respectively The HCDR3 amino acid sequence and the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 amino acid sequences of SEQ ID NOs: 9, 10 and 11 respectively, and the antibody is of the IgG1 isotype. The use according to claim 1, wherein the antibody that specifically binds to CD38 elicits an immune response in the patient. The use according to claim 2, wherein the immune response is an effector T cell (Teff) response. The use according to claim 3, wherein the Teff response is ^{mediated by CD4 +} T cells or CD8 ⁺ T cells. The use according to claim 4, wherein the Teff response is ^{mediated by the CD8 +} T cells. The use according to claim 3, wherein the Teff response is an increase in the number of ^{CD8 + T cells, an increase in CD8 +} T cell proliferation, an increase in T cell lineage expansion, an increase in CD8 ⁺ memory cell formation, and antigen dependence Increased production of sexual antibodies, increased production of cytokines, increased production of chemokines, or increased production of interleukins. The use according to claim 1, wherein the antibody that specifically binds to CD38 inhibits the function of immunosuppressive cells. The use according to claim 7, wherein the immunosuppressive cell line regulates T cells (Treg). The use according to claim 8, wherein the Treg is a CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cell. The use as described in claim 9, wherein the Treg expresses CD38. The use according to claim 10, wherein the function of the Treg is inhibited by killing the Treg. The use according to claim 11, wherein the killing of the Treg is mediated by antibody-dependent cellular cytotoxicity (ADCC). The use according to claim 7, wherein the immunosuppressive cell line is bone marrow-derived suppressor cell (MDSC). The use according to claim 13, wherein the MDSC is CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cells. The use according to claim 14, wherein the CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cells express CD38. The use according to claim 15, wherein the function of the MDSC is inhibited by killing the MDSC. The use described in claim 16, wherein the killing of MDSC is mediated by ADCC. The use according to claim 7, wherein the immunosuppressive cell line regulates B cells (Breg). The use according to claim 18, wherein the Breg is a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell. The use according to claim 19, wherein the Breg function is inhibited by killing the Breg. The use described in claim 20, wherein the killing of the Breg is mediated by ADCC. The use according to claim 7, wherein the immunosuppressive cells are present in bone marrow or in peripheral blood. The use according to claim 1, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer (stomach cancer), ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, urogenital cancer , Endometriosis, cervical cancer, or metastatic lesions of the cancer. The use according to claim 23, wherein the solid tumor lacks detectable CD38 manifestations. The use according to claim 1, wherein the antibody that specifically binds to CD38 is a non-agonistic antibody. The use according to claim 1, wherein the antibody that specifically binds to CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5. The use according to claim 23, wherein the antibody that specifically binds to CD38 comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO: 13. The use according to claim 1, wherein the antibody system that specifically binds to CD38 is administered in combination with a second therapeutic agent. The use according to claim 28, wherein the second therapeutic agent is a chemotherapeutic agent, a targeted anti-cancer therapy, a standard care drug for the treatment of solid tumors, or an immune checkpoint inhibitor. The use according to claim 29, 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. The use according to claim 30, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody. The use according to claim 31, wherein the anti-PD-1 antibody comprises a) the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 23; b) the VH of SEQ ID NO: 24 and SEQ ID NO: 25 C) the VH of SEQ ID NO: 32 and the VL of SEQ ID NO: 33; or d) the VH of SEQ ID NO: 34 and the VL of SEQ ID NO: 35. The use according to claim 30, wherein the immune checkpoint inhibitor is an anti-PD-L1 antibody. The use according to claim 33, wherein the anti-PD-L1 antibody comprises a) the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27; b) the VH of SEQ ID NO: 28 and SEQ ID NO: 29 VL; or c) the VH of SEQ ID NO: 30 and the VL of SEQ ID NO: 31. The use according to claim 30, wherein the immune checkpoint inhibitor is an anti-PD-L2 antibody. The use according to claim 30, wherein the immune checkpoint inhibitor is an anti-LAG3 antibody. The use according to claim 30, wherein the immune checkpoint inhibitor is an anti-TIM-3 antibody. The use according to claim 37, wherein the anti-TIM-3 antibody comprises a) the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 37; or b) the VH and SEQ ID NO of SEQ ID NO: 38: VL of 39. The use according to claim 28, wherein the second therapeutic agent is administered simultaneously. The use according to claim 28, wherein the second therapeutic agent is administered sequentially or separately. The use according to claim 1, wherein the antibody system that specifically binds to CD38 is administered intravenously. The use according to claim 1, wherein the antibody system that specifically binds to CD38 is administered subcutaneously in a pharmaceutical composition, the pharmaceutical composition comprising the antibody that specifically binds to CD38 and hyaluronidase. The use according to claim 1, wherein the patient is treated with radiotherapy or has been treated with radiotherapy. The use according to claim 1, wherein the patient has already undergone surgery or will undergo surgery. The use of an antibody that specifically binds to CD38 in the manufacture of a drug for inhibiting the activity of immunosuppressive cells, wherein the antibody comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, and SEQ ID NO: 6, 7 and 8, respectively The HCDR3 amino acid sequence and the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 amino acid sequences of SEQ ID NOs: 9, 10 and 11 respectively, and the antibody is of the IgG1 isotype. The use according to claim 45, wherein the immunosuppressive cell line is Treg. The use according to claim 46, wherein the Treg is a CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cell. The use according to claim 45, wherein the immunosuppressive cell line is MDSC. The use according to claim 48, wherein the MDSC is CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cells. The use according to claim 45, wherein the immunosuppressive cell line is Breg. The use according to claim 50, wherein the Breg is a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell. The use according to claim 45, wherein the antibody that specifically binds to CD38 is a non-agonistic antibody. The use according to claim 45, wherein the antibody that specifically binds to CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5. The use according to claim 45, wherein the antibody that specifically binds to CD38 comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO: 13. The use of an antibody that specifically binds to CD38 in the manufacture of a drug for enhancing the immune response of a patient, wherein the antibody comprises the heavy chain complementarity determining regions of SEQ ID NO: 6, 7, 8, 9, 10, and 11, respectively ( HCDR) 1, HCDR2, HCDR3, light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3 amino acid sequences, and the antibody is of the IgG1 isotype. The use according to claim 55, wherein the patient has cancer or viral infection. The use according to claim 55, wherein the antibody that specifically binds to CD38 is a non-agonistic antibody. The use according to claim 55, wherein the antibody that specifically binds to CD38 comprises the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5. The use according to claim 55, wherein the antibody that specifically binds to CD38 comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO: 13.

Images