Monoclonal antibodies against human MAC-1 and uses thereof

Abstract

Monoclonal antibodies against human Mac-1 are provided. These antibodies can bind to different states of Mac-1 to modify the biological functions of Mac-1. These antibodies can modulate Th1 / Th2 cytokine secretion by TLR-activated immune cells and can be used to treat diseases associated with acute and chronic inflammatory disorders, such as infectious diseases and cancer.

Description

[Technical field]

[0001] The present invention provides monoclonal antibodies against human Mac-1. These antibodies can bind to different states of Mac-1 so as to modify the biological functions of Mac-1. These antibodies can modulate Th1 / Th2 cytokine secretion by TLR-activated immune cells and can be used to treat diseases associated with acute and chronic inflammatory disorders, such as infectious diseases and cancer. [Background technology]

[0002] Macrophage-1 antigen (Mac-1, integrin αMβ2) is expressed primarily on the surface of innate immune cells (including monocytes, neutrophils, NK cells, etc.). Mac-1 is a heterodimeric glycoprotein that contains the non-covalently bound integrin αM (CD11b, CR3A, ITGAM) and integrin β2 (CD18, ITGB2). CD11b is a transmembrane protein with a large extracellular domain and a short cytoplasmic tail. The extracellular domain of CD11b contains an I domain, a β-propeller domain, a Thigh domain, a calf-1 domain, and a calf-2 domain. The I domain of CD11b has approximately 179 amino acids and is inserted into the β-propeller domain. This I domain is involved in binding to promiscuous ligands (e.g., iC3b, fibrinogen, ICAM-1, CD40L, etc.) and is involved in cell adhesion, migration, chemotaxis, and phagocytosis, regulating the inflammatory response of innate immune cells.

[0003] Like other integrins, Mac-1 exists in unique conformations with different ligand binding affinities. As shown in Figure 1, CD11b and CD18 bend into a V-shape with the I-domain close to the membrane to form inactive Mac-1 (low affinity). Inside-out signaling changes Mac-1 to an open conformation, moving the I-domain away from the membrane for optimal ligand binding. One of the epitopes located on I-EGF2 of CD18 is hidden within the bent conformation (inactive or closed state); in the extended or open state this epitope is exposed and can be recognized by a monoclonal antibody (KIM127) (J. Immunol. 2001; 166: 5629-5637). This conformational change also results in a rearrangement of the I domain site such that it becomes a high affinity site for ligand binding and forms an epitope for mAb m24 binding (Proc. Natl. Acad. Sci. USA. 2004; 101: 2333-2338). Such conformational changes, accompanied by changes in ligand binding affinity, are implicated in Mac-1 function. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO 2020 / 033929 A1 [Patent Document 2] WO 2019 / 177669 A1 [Patent Document 3] WO 2016 / 197974 A1 [Non-patent literature]

[0005] [Non-Patent Document 1] J. Immunol. 2001; 166: pp. 5629-5637 [Non-Patent Document 2] Proc.Natl.Acad.Sci.USA.2004; 101: pp. 2333~2338 [Non-Patent Document 3] Proc Natl Acad Sci USA. April 1, 2008;105(13):5195~200 [Non-Patent Document 4] Nat Immunol. August 2010;11(8):734~42 [Non-Patent Document 5] C. Orlandi et al., "A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication," J. Transl. Med. 18, 204 (2020). Doi: 10.1186 / s12967-020-02368-y Summary of the Invention [Problem to be solved by the invention]

[0006] Embodiments of the present invention relate to antibodies capable of specifically binding to Mac-1 and modulating immune cell function. These antibodies may be used to treat a variety of Mac-1-related diseases or conditions, such as infectious diseases or cancer. [Means for solving the problem]

[0007] One aspect of the present invention relates to an antibody against human Mac-1. The antibody against human Mac-1 according to one embodiment of the present invention comprises a light chain variable region sequence and a heavy chain variable region sequence selected from SEQ ID NO: 1 to SEQ ID NO: 158 shown in Table I.

[0008] One aspect of the present invention relates to a method for treating a disorder associated with Mac-1. The method according to one embodiment of the present invention comprises administering to a subject in need thereof an effective amount of an antibody of the present invention. The disorder is acute or chronic inflammation. The disorder may be an infectious disease or cancer.

[0009] Other aspects of the present invention will become apparent from the following description and associated drawings. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows two structures of Mac-1 and its epitopes for activation-sensitive mAbs. [Diagram 2] Figure 2 shows the results of characterization of HEK293 / Mac-1 using various antibodies. HEK293 cells were incubated in PBS (mock) or PBS / MnCl2 (Mn2+). Binding of isotype control IgG, CD11b-specific mAb (clone ICRF44), CD18-specific mAb (clone 6.7) or β2 activation-dependent mAbs (KIM127 and m24) was detected using flow cytometry. [Diagram 3] Figure 3A shows that representative anti-Mac-1 antibodies of the invention (DF3M-5, H4L2, m2396, 24G05 and 28E07-HH) bind primarily to myeloid immune cells (monocytes and neutrophils). Other antibodies of the invention show similar properties. Figure 3B shows that clones m2396, DF3M-5 and 24G05 bind to the murine Mac-1 expressing cell line Raw264.7. [Figure 4] FIG. 4 shows examples of anti-Mac-1 antibodies that can modulate the conformational changes of Mac-1 under PBS (mock) or PBS / MnCl2 (Mn2+) conditions. [Diagram 5] Figure 5 shows that anti-Mac-1 antibody treatment can modulate TLR4 agonist-induced Th1 / Th2 cytokine responses in mice in vivo. Data are shown as mean ± SEM (4 mice per group). [Figure 6] Figure 6 shows that anti-Mac-1 antibodies reduce tumor growth in a humanized NOG-EXL mouse model bearing A549 human lung tumors in vivo. Data are shown as mean ± SEM (10 mice per group). [Figure 7]Figure 7A shows that anti-Mac-1 antibodies enhanced the expression of functional markers in myeloid cells isolated from HIV patients, and Figure 7B shows that anti-Mac-1 antibodies reduced viral load in PBMCs from HIV patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Embodiments of the present invention relate to antibodies capable of specifically binding to Mac-1 and modulating immune cell function. These antibodies may be used to treat a variety of Mac-1-related diseases or conditions, such as infectious diseases or cancer.

[0012] A phage display library of human and mouse antibodies was constructed and screened to isolate clones carrying specific antibody genes capable of recognizing Mac-1. These anti-Mac-1 antibodies have been shown to bind Mac-1 on HEK293 / Mac-1 cells and innate immune cells. These antibodies can selectively bind to different states of Mac-1 (bent or extended / open conformation) and modulate the conformational changes of Mac-1. These anti-Mac-1 antibodies have been shown to modulate TLR-induced cytokine production and thus can be used to treat acute and chronic inflammatory disorders such as infectious diseases (see WO 2020 / 033929 A1) and cancer (see WO 2019 / 177669 A1 and WO 2016 / 197974 A1).

[0013] Specific examples of various aspects of the invention are described below, but those skilled in the art will recognize that these specific examples are merely illustrative and that other modifications and variations are possible without departing from the scope of the invention.

[0014] Materials and Methods Reagents and antibodies Antibodies and reagents used for flow cytometry were KIM127 (hybridoma from ATCC), m24-PE (BioLegend), anti-CD11b-APC (clone ICRF44, BioLegend), anti-CD18-APC (clone 6.7, BD), BSA (BioShop), rat IgG1κ-APC (BioLegend) and rat IgG1κ-PE (BioLegend). KIM127 antibody and BSA were conjugated with CF647, i.e., labeled with a CF647 labeling kit (CF Dye & Biotin SE Protein Labeling Kits, Biotium).

[0015] Cell culture and stable transfection Stable transfection of human integrin Mac-1 in HEK293 cells (BCRC) was performed using the jetPRIME® (PolyPlus) transfection protocol. Briefly, HEK293 cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM, Corning) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 50 IU / mL penicillin and streptomycin (Corning). Cells were plated at 8 × 10 5The cells were seeded at 5000 cells / well. The next day, a mixture of jetPRIME® reagent and 2 μg of pcDNA3.1 / human CD18 expression plasmid carrying a hygromycin resistance gene was added to the cells, and the cells were cultured for 24 hours. A selection antibiotic, hygromycin B (InvivoGen), was added at a concentration of 200 μg / mL, and half of the culture medium containing the antibiotic was replaced every 2 to 3 days. After 3 weeks, CD18-expressing cells were harvested using a Cell Sorter (SH800Z, Sony) to select CD18-high expressing cells, and seeded at single cell / well and 5000 cells / well in 24-well plates (Coster) and 6-well plates. The cells were maintained at 37°C in DMEM medium containing 10% heat-inactivated fetal bovine serum, 50 IU / mL penicillin and streptomycin, and 200 μg / mL hygromycin B. After the cells were enriched, human CD18 expression was analyzed by flow cytometry using anti-human CD18 APC (clone 6.7, BD) antibody. Permanent HEK293 / human CD18 cells (clone 2B4) were plated at 6 × 10 5 Cells were seeded at 100 cells / well. The transfection protocol for human CD11b was the same as above. Briefly, 2 μg of pcDNA3.1 / human CD11b plasmid was mixed with jetPRIME® reagent and added to the cells. The next day, selection antibiotics, 1 mg / mL G418 (InvivoGen) and 200 μg / mL hygromycin B, were added, and the medium containing selection antibiotics was changed every 2-3 days. Mac-1 expression was measured by flow cytometry using anti-human CD11b-APC (clone ICRF44, BioLegend) and anti-hCD18-APC. Stable clones 1-4 were selected.

[0016] Flow cytometry HEK293 / Mac-1 cells (clones 1-4) were counted and washed twice with staining buffer (PBS containing 1% FBS and 0.1% sodium azide). Cells were incubated at a concentration of 1 × 10 in staining buffer. 5The cells were adjusted to 100 cells / mL and treated with or without 0.25 mM MnCl2 (Sigma). Cells were treated with anti-Mac-1 antibody and fluorescently conjugated anti-human IgG4 antibody and incubated for 15 minutes. After washing with staining buffer, cells were analyzed by flow cytometry. In some cases, the cells were treated with antibodies (ICRF44, KIM127, m24 or isotype control) with or without 10 μg / mL anti-Mac-1 antibody. Cells were then incubated for 30 minutes at 37°C. After washing with staining buffer, cells were analyzed by flow cytometry.

[0017] Cytokine measurements Balb / c mice (n=4 / group) were intraperitoneally injected with 5mg / kg LPS and 10mg / kg anti-Mac-1 antibody for 4 hours. Mouse Th1 and Th2 cytokines in serum were detected by ProcartaPlex MS (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0018] Cancer Treatment Protocols Human umbilical cord blood-derived CD34+ cells were transplanted into NOG-EXL mice via the tail vein. Approximately 10 weeks after transplantation, peripheral blood was collected from humanized NOG-EXL animals under anesthesia and used for FACS analysis. The types, percentages, fluorescence intensity and absolute numbers of immune cells (T cells, B cells, dendritic cells and monocytes) were analyzed. The average hCD45 + %>15%, hCD45 + Of these, hCD3 + %>3%, and hCD45 + Of these, hCD14 + In cases where %>5%, humanized NOG-EXL animals were used in anti-cancer studies.

[0019] Human lung cancer A549 cells were cultured in F-12K medium with 10% FBS in a 37°C incubator containing 5% CO2. The cells were subcultured for no more than 10 passages before being inoculated into mice. A549 cells (5 × 10 6Cells (individual cells) were mixed with Matrigel (volume / volume 1:1) in a volume of 200 µL immediately prior to subcutaneous injection. Before inoculation, mice were anesthetized with 2–5% isoflurane.

[0020] Tumor volume 20-50mm 3 Once the tumor-bearing animals reach a total cell count of 100,000 cells / mL, the tumor-bearing animals are transfected with human CD45 + Frequency of macrophages in cells, human CD45 + CD3 in cells + Based on the frequency of cells and tumor volume, the mice were divided into three groups, each containing 10 mice. The day of grouping was designated as day 0. The mice were treated on day 0.

[0021] Tumor volume: Tumor volume was calculated as follows: V = (length x width) 2 ) / 2. Tumor volumes were measured and recorded twice weekly during the inoculation, grouping, and dosing periods. Tumor growth inhibition (TGI) was calculated as follows: TGI=(1-(T / C))×100%; T and C are the mean tumor volumes of the treatment and control groups, respectively, on the day of measurement.

[0022] Infectious Disease Treatment Protocols Isolation of PBMCs from HIV patients Patients were receiving regular highly active antiretroviral therapy (ART), had undetectable plasma viral load (HIV-1 RNA <50 copies / mL), and had countable CD4 cells (counts >200 cells / mm 3 Fifteen HIV-1-infected patients were recruited at National Taiwan University Hospital (Taipei, Taiwan). Clinical and laboratory data were collected and retrieved from medical records. Each blood sample was processed within 24 hours after collection, and leukocytes were isolated for further testing. The study was approved by the Institutional Review Board of National Taiwan University Hospital (Taipei, Taiwan), and written informed consent was obtained from each participant.

[0023] Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples by Ficoll-Paque (Amersham Biosciences, Sweden) gradient centrifugation. Cells were cultured in 96-well U-bottom culture plates (2 × 10 5 The cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 100 nM elvitegravir (Cayman), and 100 nM efavirenz (Cayman) in the presence of 10 μg / mL human IgG4 antibody (BioLegend) or anti-Mac-1 antibody (clone H4L2) for 3 days.

[0024] Detection of functional markers in PBMCs of HIV patients Prior to staining with fluorochrome-conjugated antibodies, cell suspensions were incubated with Fc blocker (BD Bioscience) in PBS containing 1% FBS and 0.1% sodium azide. Antibodies against CD11b (clone ICRF44, BioLegend), CD86 (clone 2331, BioLegend), HLA-DR (clone L243, BioLegend), and CD80 (clone L307, BD) were used to stain for markers. FVS786 viability stain was used to exclude dead cells from the analysis. The mean fluorescence intensity of stained cells was measured by CytoFlex flow cytometry and analyzed by Kaluza software (Beckman Coulter).

[0025] Detection of intracellular HIV virus - Quantification of two long terminal repeat (LTR)-DNA circles PBMCs (3 × 10 in 24-well culture plates) were cultured for 3 days in the presence of human IgG4 antibody (BioLegend) or anti-Mac-1 antibody (H4L2, 10 μg / mL) with or without treatment with PMA (100 ng / mL) and ionomycin (1 μg / mL). 6DNA from 1000 cells / well was extracted with a QIAamp DNA Blood Mini Kit (Qiagen, MD, USA) and DNA was eluted with 50 μL of nuclease-free water. Digital PCR was performed using the QX100™ Droplet Digital™ PCR platform (Bio-Rad, Hercules, CA). ddPCR mixtures were made by adding 1-5 μL of sample to 10 μL of 2×ddPCR™ supermix for probes (Bio-Rad), 1 μL of EcoR, 500 nM primers and 250 nM probe in a final volume of 20 μL. The mixture was placed in an 8-channel cartridge, 70 μL of droplet generation oil (Bio-Rad) was added, and droplets were generated in a QX100™ droplet generator (Bio-Rad). Droplets in oil suspension were transferred to a ddPCR 96-well plate (Bio-Rad) and PCR was performed in a T100™ Thermal Cycler (Bio-Rad). The ddPCR amplification reaction consisted of an initial denaturation at 95° C. for 10 min, followed by 40 cycles of denaturation at 95° C. for 15 s and annealing / extension at 60° C. for 60 s, and enzyme inactivation at 98° C. for 10 min. The ramp rate for each step was 2° C. / s. The sequences of the primer pairs are listed in Table VI.

[0026] statistical analysis Results were compared by Fisher's exact test for categorical variables and by paired or unpaired t-tests, as appropriate, for continuous variables. Data are reported as mean ± SEM. Statistical analysis was performed using Prism 9.0 software. Two-tailed tests were used and a p-value <0.05 was considered statistically significant.

[0027] result Expression of the heterodimer CD11b / CD18 (Mac-1) on the surface of HEK293 cells HEK293 cells that do not express endogenous Mac-1 were transfected with pcDNA3.1 / human CD11b and pcDNA3.1 / human CD18 plasmids using liposomal transfection. After G418 and hygromycin selection, several single cell clones stably expressing human Mac-1 on the cell surface were obtained by FACS sorting using CD18-specific mAb (clone 6.7) and CD11b-specific mAb (clone ICRF44). One clone (designated 1-4) was selected for all examples shown in this description. Other clones show similar properties.

[0028] Expression of CD11b and CD18 on HEK293 cells detected by flow cytometry is shown in Figure 2. Two activation-sensitive antibody mAbs, KIM127 and m24, were used to examine the conformational state on the surface of HEK293 / Mac-1 cells. KIM127 could bind well to HEK293 / Mac-1, suggesting that Mac-1 is in an extended conformation. Low binding of m24 to HEK293 / Mac-1 cells was observed in PBS (mock), suggesting that Mac-1 is in a low affinity state. In PBS, HEK293 / Mac-1 is partially activated. In contrast, Mn 2+ Treatment induced 100% of the Mac-1 molecules to adopt the extended high affinity structure, thus providing an excellent platform for screening monoclonal antibodies against human Mac-1.

[0029] Anti-Mac-1 antibodies selectively bind to different states of Mac-1 on the surface of HEK293 / Mac-1 cells. Phage display libraries of human and mouse antibodies were constructed and then screened to isolate clones carrying specific antibody genes capable of recognizing Mac-1. A total of 79 clones were selected from the phage pools in each round of selection. The amino acid sequences of the variable regions of these clones are listed in Table I and Table VII. To verify that these clones can bind to Mac-1 in their native conformation, recombinant antibodies were generated from these clones with human IgG4 backbone. The recombinant anti-Mac-1 antibodies were used for flow cytometric analysis of HEK293 / Mac-1 cells. As listed in Table II, these antibodies can indeed recognize Mac-1 on the surface of HEK293 / Mac-1 cells.

[0030] The conformational changes of Mac-1 are involved in the regulation of its function. We examined whether these antibodies could recognize different conformations of Mac-1. As listed in Table II, some clones were found to be related to the Mn 2+ The clones selectively bind to activation-specific epitopes on the Mac-1 molecule in HEK293 / Mac-1 cells after stimulation with MnCl2 (mock / MnCl2 ratio <1). In contrast, some clones preferentially recognize the resting form of Mac-1 (mock / MnCl2 ratio >1). The predicted amino acid sequences of the CDR and framework regions of the selected clones are shown in Table I.

[0031] Anti-Mac-1 antibodies bind primarily to Mac-1 on the surface of innate immune cells Monocytes (CD14 + cells) and neutrophils (CD66b + Innate immune cells such as CD14+ cells are the major cells expressing Mac-1 on their cell surface. A subpopulation of B cells also expressed Mac-1 on their cell surface (Proc Natl Acad Sci USA. 2008 Apr. 1;105(13):5195-200). The specificity of selective anti-Mac-1 antibodies was determined by flow cytometry using human whole blood. As shown in Figure 3A, the anti-Mac-1 antibodies in this example bound to innate immune cells (CD14+ cells). +and CD66b + cells) and B cells (CD19 + In contrast, these antibodies were able to bind to a small population of T cells (CD3 + Lymphocytes). Taken together, these results indicate that anti-Mac-1 antibodies can specifically bind to Mac-1 epitopes on immune cells. To determine whether anti-Mac-1 antibodies validated against human Mac-1 will cross-react with mouse Mac-1, the Raw 264.7 mouse macrophage cell line expressing mouse Mac-1 on the cell surface is used. As shown in Figure 3B, some clones such as DF3M-5, m2396 and 24G05 can bind to the surface of Raw 264.7, suggesting that these clones can cross-react with mouse Mac-1.

[0032] Anti-Mac-1 antibodies induce conformational changes in Mac-1 It is well known that inside-out signaling induces a global conformational change in Mac-1 resulting in outside-in signaling. To screen which anti-Mac-1 antibodies induce conformational changes in Mac-1, we used KIM127 and m24 antibodies, which preferentially bind to the extended conformation of Mac-1, as reporters to detect conformational changes. As shown in Figure 4, left panel, incubation of HEK293 / Mac-1 cells with the antibodies in PBS buffer (mock) resulted in basal levels of KIM127 and m24 binding. In contrast, incubation of HEK293 / Mac-1 cells in MnCl2 / PBS buffer (Mn 2+Incubation of HEK293 / Mac-1 cells with the antibody in simulated conditions induced maximal levels of KIM127 and m24 binding (Figure 4, right panel). Incubation with DF3M-5 in simulated conditions induced a small increase in KIM127 binding and a large increase in m24 binding (Figure 4, left panel), indicating that this DF3M-5 clone may function as an agonist to enhance the conformational change of Mac-1. Other clones that may function as agonists (based on m24 relative expression >1 to IgG4 control) are listed in Table III. In contrast, Mn 2+ Incubation with 28E07 under conditions induced a small decrease in KIM127 binding and a large decrease in m24 binding (Figure 4, right), indicating that this 28E07 clone may function as an antagonist to reduce conformational changes in Mac-1. Other clones that may function as antagonists (based on m24 relative representation to IgG4 control <1) are listed in Table IV. The results of these studies indicate that the antibodies of the invention may be used selectively to control conformational changes in Mac-1 and thereby modulate Mac-1 function.

[0033] Anti-Mac-1 antibodies modulate Th1 / Th2 cytokine secretion by TLR-activated immune cells in vivo Previous studies have shown that active CD11b integrin is involved in crosstalk with MyD88 and TRIF pathways and modulates TLR signaling in innate immune responses (Nat Immunol. 2010 Aug;11(8):734-42). To test whether the anti-Mac-1 antibody of the present invention can modulate Th1 / Th2 cytokine secretion in TLR-activated immune cells in vivo, Balb / c mice (n=4 / group) were intraperitoneally injected with 5mg / kg LPS and 10mg / kg anti-Mac-1 antibody. After 4 hours, serum Th1 / Th2 cytokines were measured by ProcartaPlex™ MS. As shown in FIG. 5, m2396 and DF3M-5 treatment enhance TLR4-induced Th1 cytokines (such as IFN-γ, IL-1β, and TNF-α) in serum and slightly enhance TLR4-induced Th2 cytokines (such as IL-5 and IL-13). These results suggest that anti-Mac-1 antibody (clone m2396 and DF3M-5) treatment can skew the TLR-induced Th1 / Th2 response. In contrast, 24G05 treatment did not alter the Th1 / Th2 cytokine profile.

[0034] Anti-Mac-1 antibody treatment reduces tumor growth The anti-cancer activity of anti-Mac-1 antibodies of the invention (eg, m2396 and 28E07-HH) was further evaluated in treating the A549 cancer model in female NOG-EXL humanized mice.

[0035] The mean tumor volume was approximately 41 mm 3 Once the tumor-bearing mice reached the target tumor mass index (MMI), they were randomized into three groups (human IgG4, m2396, and 28E07-HH) and treatment was initiated. Thirty-five days after grouping, the average tumor size in mice was 172.59 mm in the human IgG4 group and 172.59 mm in the human IgG4 group. 3 , 132.51mm in m2396 group 3 and 109.88 mm in the 28E07-HH group. 3(Figure 6). Figure 6 shows the results of representative antibodies m2396 and 28E07-HH. Other antibodies of the present invention have similar properties. The tumor growth inhibition (TGI)% of the m2396 group and the 28E07-HH group was 23.59% and 35.93%, respectively. The TGI of different groups at different time points was shown in Table V. These results indicate that these anti-Mac-1 antibodies can function as therapeutic antibodies for treating human cancer.

[0036] Anti-Mac-1 antibody treatment in HIV patients reduces HIV viral load and reverses the immunosuppressive phenotype of PBMCs To test the effectiveness of the inhibitory effect mediated by anti-Mac-1 antibodies against HIV, PBMCs isolated from 15 latently HIV-infected patients were treated with anti-Mac-1 antibodies in vitro for 3 days. As shown in FIG. 7A, anti-Mac-1 antibodies (H4L2 shown as a representative example of anti-Mac-1 antibodies) significantly enhanced the expression of functional markers CD86 and MHC class II in myeloid cells of HIV patients. These results indicate that enhanced T cell activation and maturation of dendritic cells (DCs) in HIV patients can be achieved with the anti-Mac-1 antibodies of the present invention. These enhanced immune responses suggest that the antibodies of the present invention may potentially be applied in the treatment of HIV infection.

[0037] Although combination antiretroviral therapy (ART) can suppress HIV replication, HIV-1 persists in infected cells as a stably integrated genome, as well as less stable unintegrated DNA, including linear, 1-LTR and 2-LTR circular DNA. Although less abundant, 2-LTR circular DNA is considered a surrogate marker of recent infection events and is currently used as a diagnostic tool. (C. Orlandi et al., "A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication," J. Transl. Med. 18, 204 (2020). Doi: 10.1186 / s12967-020-02368-y).

[0038] To detect the amount of intracellular HIV virus, the HIV viral DNA reservoir was quantified using two long terminal repeat (LTR) DNA circles as markers. Because these 15 HIV-1-infected patients were receiving regular highly active antiretroviral therapy (ART) treatment, only three of the 15 patients had detectable levels of HIV DNA by LTR assay. Nevertheless, a decline in HIV 2LTR DNA levels was observed in PBMC samples from these three patients treated with anti-Mac-1 antibody or with a combination of anti-Mac-1 antibody and phorbol 12-myristate 13-acetate (PMA) and ionomycin (Figure 7B).

[0039] While the present invention has been described with a limited number of examples, those skilled in the art will recognize that these examples are merely illustrative and that other modifications and variations are possible without departing from the scope of the invention. Accordingly, the scope of protection should be limited only by the appended claims.

[0040] [Table 1A]

[0041]

Table 1B

[0042]

Table 1C

[0043]

Table 1D

[0044]

Table 1E

[0045]

Table 1F

[0046]

Table 1G

[0047]

Table 1H

[0048]

Table 1I

[0049]

Table 1J

[0050]

Table 1K

[0051]

Table 1L

[0052]

Table 1M

[0053]

Table 1N

[0054]

Table 1O

[0055]

Table 1P

[0056]

Table 1Q

[0057]

Table 1R

[0058]

Table 1S

[0059]

Table 1T

[0060]

Table 1U

[0061]

Table 1V

[0062]

Table 1W

[0063]

Table 1X

[0064]

Table 1Y

[0065]

Table 1Z

[0066]

Table 1AA

[0067]

Table 2

[0068]

Table 3

[0069]

Table 4

[0070]

Table 5

[0071]

Table 6

[0072]

Table 7A

[0073]

Table 7B

[0074]

Table 7C

[0075]

Table 7D

[0076]

Table 7E

[0077]

Table 7F

[0078]

Table 7G

[0079]

Table 7H

[0080]

Table 7I

[0081]

Table 7J

[0082]

Table 7K

[0083]

Table 7L

[0084]

Table 7M

[0085]

Table 7N

[0086]

Table 7O

[0087]

Table 7P

[0088]

Table 7Q

[0089]

Table 7R

[0090]

Table 7S

[0091]

Table 7T

[0092]

Table 7U

[0093]

Table 7V

Claims

1. An antibody against human Mac-1, which binds to a specific state of human Mac-1 and modulates Th1 and / or Th2 cytokine secretion by TLR-activated immune cells.

2. The antibody of claim 1, wherein the Th1 cytokine secretion is enhanced to a greater extent than Th2 cytokine secretion.

3. the antibody comprises a heavy chain variable region sequence having CDR-H1, CDR-H2, and CDR-H3 sequences; and a light chain variable region sequence having CDR-L1, CDR-L2, and CDR-L3 sequences; The CDR-H1 is selected from SEQ ID NOs: 543, 549, 483, 369, 189, 159, 165, 171, 177, 183, 195, 201, 207, 213, 219, 225, 231, 237, 243, 249, 255, 261, 267, 273, 279, 285, 291, 297, 303, 309, 315, 321, 330, 332, 334, 336, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, having the sequence of 27, 333, 339, 345, 351, 357, 363, 375, 381, 387, 393, 399, 405, 411, 417, 423, 429, 435, 441, 447, 453, 459, 465, 471, 477, 489, 495, 501, 507, 513, 519, 525, 531, or 537; The CDR-H2 is selected from SEQ ID NOs: 544, 550, 484, 370, 190, 160, 166, 172, 178, 184, 196, 202, 208, 214, 220, 226, 232, 238, 244, 250, 256, 262, 268, 274, 280, 286, 292, 298, 304, 310, 316, 322, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 349, 350, 356 ... having the sequence of 28, 334, 340, 346, 352, 358, 364, 376, 382, ​​388, 394, 400, 406, 412, 418, 424, 430, 436, 442, 448, 454, 460, 466, 472, 478, 490, 496, 502, 508, 514, 520, 526, 532, or 538; The CDR-H3 is selected from SEQ ID NOs: 545, 551, 485, 371, 191, 161, 167, 173, 179, 185, 197, 203, 209, 215, 221, 227, 233, 239, 245, 251, 257, 263, 269, 275, 281, 287, 293, 299, 305, 311, 317, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 29, 335, 341, 347, 353, 359, 365, 377, 383, 389, 395, 401, 407, 413, 419, 425, 431, 437, 443, 449, 455, 461, 467, 473, 479, 491, 497, 503, 509, 515, 521, 527, 533, or 539; The CDR-L1 is selected from the group consisting of SEQ ID NOs: 546, 552, 486, 372, 192, 162, 168, 174, 180, 186, 198, 204, 210, 216, 222, 228, 234, 240, 246, 252, 258, 264, 270, 276, 282, 288, 294, 300, 306, 312, 318, 324, 330, 332, 334, 336, 338, 339, 340, 342, 346, 348, 349, 350, 352, 356, 358, 360, 362, 364, 366, 368, 370, 376, 378, 379, 380, 382, ​​384, 386, 388, 389, 390, 392, 394, 396, 398, 400, 400, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 425, 426, 427, 428, 430, 4 having the sequence of 30, 336, 342, 348, 354, 360, 366, 378, 384, 390, 396, 402, 408, 414, 420, 426, 432, 438, 444, 450, 456, 462, 468, 474, 480, 492, 498, 504, 510, 516, 522, 528, 534, or 540; The CDR-L2 is selected from SEQ ID NOs: 547, 553, 487, 373, 193, 163, 169, 175, 181, 187, 199, 205, 211, 217, 223, 229, 235, 241, 247, 253, 259, 265, 271, 277, 283, 289, 295, 301, 307, 313, 319, 325, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, having the sequence of 31, 337, 343, 349, 355, 361, 367, 379, 385, 391, 397, 403, 409, 415, 421, 427, 433, 439, 445, 451, 457, 463, 469, 475, 481, 493, 499, 505, 511, 517, 523, 529, 535, or 541; The CDR-L3 is selected from SEQ ID NOs: 548, 554, 488, 374, 194, 164, 170, 176, 182, 188, 200, 206, 212, 218, 224, 230, 236, 242, 248, 254, 260, 266, 272, 278, 284, 290, 296, 302, 308, 314, 320, 326, 332, 338, 3. The antibody of claim 1 or 2 having the sequence: 344, 350, 356, 362, 368, 380, 386, 392, 398, 404, 410, 416, 422, 428, 434, 440, 446, 452, 458, 464, 470, 476, 482, 494, 500, 506, 512, 518, 524, 530, 536, or 542.

4. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are selected from the group consisting of SEQ ID NOs: 159-164, or SEQ ID NOs: 165-170, or SEQ ID NOs: 171-176, or SEQ ID NOs: 177-182, or SEQ ID NOs: 183-188, or SEQ ID NOs: 189-194, or SEQ ID NOs: 195-200, or SEQ ID NOs: 201-206, or SEQ ID NOs: 207-212, or SEQ ID NOs: 213-218, or SEQ ID NOs: 219-224, or SEQ ID NOs: 225-230, or SEQ ID NOs: 231-236, or SEQ ID NOs: 237-242, or SEQ ID NOs: Nos. 243-248, or SEQ ID NOs. 249-254, or SEQ ID NOs. 255-260, or SEQ ID NOs. 261-266, or SEQ ID NOs. 267-272, or SEQ ID NOs. 273-278, or SEQ ID NOs. 279-284, or SEQ ID NOs. 285-290, or SEQ ID NOs. 291-296, or SEQ ID NOs. 297-302, or SEQ ID NOs. 303-308, or SEQ ID NOs. 309-314, or SEQ ID NOs. 315-320, or SEQ ID NOs. 321-326, or SEQ ID NOs. 327-332, or SEQ ID NOs. 333-338, or SEQ ID NOs. 339-344, or SEQ ID NOs. 345- 350, or SEQ ID NOs: 351 to 356, or SEQ ID NOs: 357 to 362, or SEQ ID NOs: 363 to 368, or SEQ ID NOs: 369 to 374, or SEQ ID NOs: 375 to 380, or SEQ ID NOs: 381 to 386, or SEQ ID NOs: 387 to 392, or SEQ ID NOs: 393 to 398, or SEQ ID NOs: 399 to 404, or SEQ ID NOs: 405 to 410, or SEQ ID NOs: 411 to 416, or SEQ ID NOs: 417 to 422, or SEQ ID NOs: 423 to 428, or SEQ ID NOs: 429 to 434, or SEQ ID NOs: 435 to 440, or SEQ ID NOs: 441 to 446, or SEQ ID NOs: 447 to 452, or has the sequence of SEQ ID NO:453-458, or SEQ ID NO:459-464, or SEQ ID NO:465-470, or SEQ ID NO:471-476, or SEQ ID NO:477-482, or SEQ ID NO:483-488, or SEQ ID NO:489-494, or SEQ ID NO:495-500, or SEQ ID NO:501-506, or SEQ ID NO:507-512, or SEQ ID NO:513-518, or SEQ ID NO:519-524, or SEQ ID NO:525-530, or SEQ ID NO:531-536, or SEQ ID NO:537-542, or SEQ ID NO:543-548, or SEQ ID NO:549-554,An antibody against human Mac-1 according to any one of claims 1 to 3.

5. An antibody against human Mac-1 according to any one of claims 1 to 4, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 have the sequences of SEQ ID NO: 543 to 548, or SEQ ID NO: 549 to 554, or SEQ ID NO: 483 to 488, or SEQ ID NO: 369 to 374, or SEQ ID NO: 189 to 194.

6. The heavy chain variable region sequences are set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 9 3, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, or 157; ,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 6. An antibody against human Mac-1 according to any one of claims 1 to 5, which is 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158.

7. The heavy chain variable region sequences and the light chain variable region sequences are the following sequence pairs: SEQ ID NO:1 and 2, or SEQ ID NO:3 and 4, or SEQ ID NO:5 and 6, or SEQ ID NO:7 and 8, or SEQ ID NO:9 and 10, or SEQ ID NO:11 and 12, or SEQ ID NO:13 and 14, or SEQ ID NO:15 and 16, or SEQ ID NO:17 and 18, or SEQ ID NO:19 and 20, or SEQ ID NO:21 and 22, or SEQ ID NO:23 and 24, or SEQ ID NO:25 and 26, or SEQ ID NO:27 and 28, or SEQ ID NO:29 and 30, or SEQ ID NO:31 and 32, or SEQ ID NO:33 and 34, or SEQ ID NO:35 and 36. or SEQ ID NO:37 and 38, or SEQ ID NO:39 and 40, or SEQ ID NO:41 and 42, or SEQ ID NO:43 and 44, or SEQ ID NO:45 and 46, or SEQ ID NO:47 and 48, or SEQ ID NO:49 and 50, or SEQ ID NO:51 and 52, or SEQ ID NO:53 and 54, or SEQ ID NO:55 and 56, or SEQ ID NO:57 and 58, or SEQ ID NO:59 and 60, or SEQ ID NO:61 and 62, or SEQ ID NO:63 and 64, or SEQ ID NO:65 and 66, or SEQ ID NO:67 and 68, or SEQ ID NO:69 and 70, or SEQ ID NO:71 and 72, or SEQ ID NO:73 and 74, or or SEQ ID NO: 75 and 76, or SEQ ID NO: 77 and 78, or SEQ ID NO: 79 and 80, or SEQ ID NO: 81 and 82, or SEQ ID NO: 83 and 84, or SEQ ID NO: 85 and 86, or SEQ ID NO: 87 and 88, or SEQ ID NO: 89 and 90, or SEQ ID NO: 91 and 92, or SEQ ID NO: 93 and 94, or SEQ ID NO: 95 and 96, or SEQ ID NO: 97 and 98, or SEQ ID NO: 99 and 100, or SEQ ID NO: 101 and 102, or SEQ ID NO: 103 and 104, or SEQ ID NO: 105 and 106, or SEQ ID NO: 107 and 108, or SEQ ID NO: 109 and 110, or SEQ ID NO: Nos. 111 and 112, or SEQ ID NOs. 113 and 114, or SEQ ID NOs. 115 and 116, or SEQ ID NOs. 117 and 118, or SEQ ID NOs. 119 and 120, or SEQ ID NOs. 121 and 122, or SEQ ID NOs. 123 and 124, or SEQ ID NOs. 125 and 126, or SEQ ID NOs. 127 and 128, or SEQ ID NOs. 129 and 130, or SEQ ID NOs. 131 and 132, or SEQ ID NOs. 133 and 134, or SEQ ID NOs. 135 and 136, or SEQ ID NOs. 137 and 138, or SEQ ID NOs. 139 and 140, or SEQ ID NOs. 141 and 142, or SEQ ID NOs. 143 and 144,or SEQ ID NO: 145 and 146, or SEQ ID NO: 147 and 148, or SEQ ID NO: 149 and 150, or SEQ ID NO: 151 and 152, or SEQ ID NO: 153 and 154, or SEQ ID NO: 155 and 156, or SEQ ID NO: 157 and 158.

8. An antibody against human Mac-1 described in any one of claims 1 to 7, wherein the heavy chain variable region sequence and the light chain variable region sequence have the following sequence pairs: SEQ ID NOs: 129 and 130, or SEQ ID NOs: 131 and 132, or SEQ ID NOs: 133 and 134, or SEQ ID NOs: 71 and 72, or SEQ ID NOs: 11 and 12.

9. A pharmaceutical composition for treating a disease or disorder, wherein the disease or disorder is an infectious disease, inflammation or cancer, the pharmaceutical composition comprising an antibody described in any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient, and optionally, the infectious disease is HIV infection, the inflammation is acute and chronic inflammation, and the cancer is lung cancer.

10. A pharmaceutical composition for modulating an immune response in a subject in need thereof, comprising an antibody described in any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.

11. The pharmaceutical composition described in claim 10, wherein the immune response comprises modulated T cell activation or dendritic cell maturation.

12. The pharmaceutical composition described in claim 10, wherein the immune response is an innate immune response.

13. A pharmaceutical composition for inducing cytokine secretion by TLR-activated immune cells, the pharmaceutical composition comprising an antibody described in any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.

14. A pharmaceutical composition for inhibiting tumor growth, comprising an antibody according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.

15. A pharmaceutical composition for reducing viral load in a subject in need thereof, the pharmaceutical composition comprising an antibody described in any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.