MONOCLONAL ANTIBODY AGAINST HUMAN MAC-1 AND USES THEREOF

BACKGROUND OF INVENTION

[0001] Macrophage-1 antigen (Mac-1, integrin aM02) is mainly expressed on the surface of innate immune cells (including monocytes, neutrophils, NK cells, etc.). Mac-1 is a heterodimeric glycoprotein comprising non-covalently linked integrin aM (CD 11b, CR3A, ITGAM) and integrin 02 (CD18, ITGB2). CD1 lb is a transmembrane protein with alarge extracellular domain and a short cytoplasmic tail. Its extracellular domain comprises an I domain, a 0-propeller domain, a thigh domain, a calf-1 domain, and a calf-2 domain. The I domain of CDllb has around 179 amino acids inserting into the 0-propeller domain. This I domain is responsible for binding to promiscuous ligands (e.g., iC3b, fibrinogen, ICAM-1, CD40L, etc.) and participates in cell adhesion, migration, chemotaxis, and phagocytosis, and regulates inflammatory responses of innate immune cells.

[0002] Like other integrins, Mac-1 exists in distinct conformations with different ligand binding affinities. As shown in FIG 1, CD1 lb and CD18 are bent in a V shape with the I-domain close to the membrane to form an inactive Mac-1 (low affinity). Inside-out signaling changes the Mac-1 to an open conformation, extending the I domain away from the membrane for optimal ligand binding. One epitope located on the I-EGF2 of CD 18 is hidden in the bent conformation (inactive or closed state); this epitope becomes exposed and can be recognized by a monoclonal antibody (KIM127) in the extended or open state (J. Immunol. 2001; 166: 5629-5637). This conformational change also results in the 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 accompanying ligand binding affinity changes are tied to Mac-1 functions.

SUMMARY OF THE INVENTION

[0003] Embodiments of the invention relate to antibodies that can bind specifically to Mac-1 and modulate immune cell functions. These antibodies may be used to treat various Mac-1 associated diseases or conditions, such as infectious diseases or cancers.

[0004] One aspect of the invention relates to antibodies against human Mac-1. An antibody against human Mac-1 in accordance with one embodiment of the invention comprises a lightchain variable region sequence and a heavy -chain variable region sequence selected from SEQ ID NO:1 through SEQ ID NO: 158 shown in Table I. [0005] One aspect of the invention relates to methods for treating a disorder associate with Mac-1. A method in accordance with one embodiment of the invention comprises administering to a subject in need thereof an effective amount of an antibody of the invention. The disorder is an acute or chronic inflammation. The disorder may be an infection or a cancer.

[0006] Other aspects of the invention would become apparent from the following description and the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1 shows the two conformations of Mac-1 and their epitopes for activationsensitive mAbs.

[0008] FIG. 2 shows results of characterization of HEK293/Mac-1 using various antibodies. HEK293 cells were incubated in PBS (Mock) or PBS/MnCh (Mn ²⁺ ). Bindings of isotype control IgG, a CD1 lb specific mAb (clone ICRF44), a CD18 specific mAb (clone 6.7), or P2 activationdependent mAbs (KIM127 and m24) were detected using flow cytometry.

[0009] FIG. 3A shows that representative anti-Mac-1 antibodies of the invention (DF3M-5, H4L2, m2396, 24G05, and 28E07-HH) predominantly bind to myeloid immune cells (monocytes and neutrophils). Other antibodies of the invention show similar properties.

[0010] FIG. 3B shows that clones m2396, DF3M-5, and 24G05 bind to mouse Mac-1 expressing cell line Raw264.7.

[0011] FIG. 4 shows examples of anti-Mac-1 antibodies that can modulate conformational changes of Mac-1 under PBS (Mock) or PBS/MnCh (Mn ²⁺ ) conditions.

[0012] FIG. 5 shows that anti -Mac- 1 antibody treatments can modulate TLR4 agonist- induced Thl/Th2 cytokines responses in mice in vivo. Data are shown as the means ± SEM (4 mice per group).

[0013] FIG. 6 shows that anti-Mac-1 antibodies reduce tumor growths in A549 human lung tumor bearing humanized NOG-EXL mouse model in vivo. Data are shown as the means ± SEM (10 mice per group).

[0014] FIG. 7A shows that anti-Mac-1 antibody enhanced the expression of functional markers in myeloid cells isolated from HIV patients.

[0015] FIG. 7B shows that anti -Mac- 1 antibody reduced the virus load in PBMCs from HIV patients. DETAILED DESCRIPTION

[0016] Embodiments of the invention relate to antibodies that can bind specifically to Mac-1 and modulate immune cell functions. These antibodies may be used to treat various Mac-1 associated diseases or conditions, such as infectious diseases or cancers.

[0017] Human antibody and mouse antibody phage display libraries were constructed and screened to isolate clones carrying specific antibody genes that can recognize Mac-1. These anti- Mac-1 antibodies are shown to bind Mac-1 on the 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 are shown to modulate TLR-induced cytokine productions and therefore can be used to treat acute and chronic inflammatory disorders, such as infectious diseases (ref: WO 2020/033929 Al) and cancers (ref: WO 2019/177669 Al and WO 2016/197974 Al).

[0018] The following describes specific examples of various aspects of the invention. One skilled in the art would appreciate that these specific examples are for illustration only and that other modifications and variations are possible without departing from the scope of the invention.

Material and method

Reagents and antibodies

[0019] The antibodies and reagents used for flow cytometry are KIM 127 (hybridoma from ATCC), m24-PE (BioLegend), anti-CDl lb-APC (clone ICRF44, BioLegend), anti-CD18-APC (clone 6.7, BD), BSA (BioShop), Rat IgGlK-APC (BioLegend), and Rat IgGlK-PE (BioLegend). The KIM127 antibody and BSA were conjugated with CF647, i.e., labeled with CF647 labeling kit (CF Dye & Biotin SE Protein Labeling Kits, Biotium).

Cell culture and stable transfection

[0020] Stable transfection of human integrin Mac-1 in HEK293 cells (BCRC) was performed using jetP RIME® (PolyPlus) transfection protocols. Briefly, HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Coming), supplemented with 10% heat- inactivated fetal bovine serum (Gibco) and 50 lU/mL penicillin and streptomycin (Coming) at 37 °C. The cells were seeded at 8 x 10 ⁵ cells/well on a 6-well plate (Coster). Next day, a mixture of the jetPRIME® reagent and 2 pg pcDN A3.1 /human CD18 expression plasmid carrying a hygromycin-resistance gene was added to the cells, and the cells were cultured for 24 hr. The selection antibiotic, hygromycin B (InvivoGen), was added at a concentration of 200 pg/ml, and half of the culture media containing the antibiotic were changed every 2 to 3 days. After 3 weeks, the CD18-expression cells were collected using the Cell Sorter (SH800Z, SONY) to pick up CD18-high expression cells and seeded at single cell/well and 5000 cells/well on a24-well plate (Coster) and a 6-well plate. Cells were maintained in DMEM medium with 10% heat-inactivated fetal bovine serum, 50 lU/mL penicillin and streptomycin, and 200 pg/ml Hygromycin B at 37 °C. After the cells were enriched, human CD18 expression was analyzed with anti-human CD18- APC (clone 6.7, BD) antibody by flow cytometry. The permanent HEK293/human CD18 cells (clone 2B4) were seeded at 6 x 10 ⁵ cells/well on a 6-well plate. The transfection protocol for human CDllb was the same as above. Briefly, 2 pg pcDN A3.1 /human CDl lb plasmid was mixed with jetPRIME® reagent and added to cells. Next days, the selection antibiotics, 1 mg/ml G418 (InvivoGen) and 200 pg/ml Hygromycin B, were added, and the medium containing selection antibiotics was changed every 2 to 3 days. The Mac-1 expression was measured with anti-human CDllb-APC (clone ICRF44, BioLegend) and anti-hCD18-APC by flow cytometry. The stable clone 1-4 was picked up.

Flow cytometry

[0021] The HEK293/Mac-1 cells (clonel-4) were counted and washed twice with staining buffer (PBS containing 1% FBS, and 0.1% sodium azide). Cells were adjusted at a concentration of 1x10 ⁵ cells/ml in staining buffer and treated with/without 0.25 mM MnCh (Sigma). Cells were treated with anti -Mac- 1 antibodies and fluorescent conjugated anti -human IgG4 antibodies and incubated for 15 mins. After washing with the staining buffer, the cells were analyzed by flow cytometry. In some examples, these cells were treated with the antibodies (ICRF44, KIM127, m24, or isotype control) in the presence or absence lOpg/ml anti-Mac-1 antibodies. The cells were then incubated at 37 °C for 30 min. After washing with the staining buffer, the cells were analyzed by flow cytometry.

Cytokine measurement

[0022] Balb/c mice (n=4/group) were intraperitoneally injected with 5 mg/kg LPS and 10 mg/kg anti -Mac- 1 antibodies for 4 hours. Murine Thl and Th2 cytokines in the serum were detected by ProcartaPlex MS (Thermo Fisher Scientific) according to the manufacturer instructions.

Protocol of cancer treatment

[0023] Human umbilical cord blood derived CD34+ cells were transplanted into NOG-EXL mice via the tail vein. About 10 weeks after transplantation, peripheral blood were collected from the humanized NOG-EXL animals under anesthesia and used for FACS analysis. The types, proportions, fluorescence intensities, and absolute counts of immune cells (T cells, B cells, dendritic cells, and monocyte cells) were analyzed. When the average hCD45 ⁺ % > 15%, hCD3 ⁺ of hCD45 ⁺ % > 3%, and hCD14 ⁺ of hCD45 ⁺ % > 5%, the humanized NOG-EXL animals were used for the anti -cancer study.

[0024] Human lung cancer A549 cells were cultured in a 37 °C incubator containing 5% CO2 with 10% FBS in F-12K medium. The cells were sub-cultured within 10 passages before being inoculated into mice. A549 cells (5xl0 ⁶ cells) were mixed with Matrigel (v/v 1:1) in a volume of 200 pl immediately before injection subcutaneously. Before inoculation, mice were anesthetized with 2-5% isoflurane.

[0025] When tumor volumes reached 20 - 50 mm ³ , tumor-bearing animals were grouped into 3 groups based on the frequency of macrophage in human CD45 ⁺ cells, the frequency of CD3 ⁺ cell in human CD45 ⁺ cells, and tumor volumes, each group contains 10 mice. The day of grouping was denoted as day 0. Mice were treated on day 0.

[0026] Tumor volume: The tumor volume was calculated as follows: V = (length x width ² ) / 2. Tumor volume was measured and recorded twice a week during inoculation, grouping, and during the dose period. Tumor growth inhibition (TGI) was calculated as follows: TGI= (1- (T/C))xl00%; T and C as the mean tumor volumes of the treatment and control groups, respectively, on the measurement day.

Protocol of infectious disease treatment

PBMC isolation from HIV patients

[0027] Fifteen HIV-1 infected patients receiving regular highly active antiretroviral therapy (ART) treatments with undetectable plasma viral load (<50 HIV-1 RNA copies/ml) and countable CD4 cells (count > 200/mm ³ ) were recruited at National Taiwan University Hospital (Taipei, Taiwan). The clinical and laboratory data were collected and acquired from medical records. Each blood sample was processed within 24 hours after collection, and leukocytes were isolated for further examination. This study was approved by the Institutional Review Board of National Taiwan University Hospital (Taipei, Taiwan), and written informed consents were obtained from each participant.

[0028] Peripheral blood mononuclear cells (PBMC) were isolated from whole blood samples by means of Ficoll-Paque (Amersham Biosciences, Sweden) gradient centrifugation. Cells were cultured in 96-well U-bottom culture plates (2x10 ⁵ cells/well) and resuspended in RPMI-1640 medium with 10% fetal bovine serum (FBS), 100 nM elvitegravir (Cayman), and 100 nM efavirenz (Cayman) in the presence of human IgG4 antibody (BioLegend) or anti -Mac- 1 antibody (clone H4L2) 10 pg/ml for 3 days.

Functional marker detection of PBMCs of HIV patients

[0029] Cell suspensions were incubated with Fc blocker (BD Bioscience) in PBS containing 1% FBS and 0.1% sodium azide before staining with fluorochrome-labeled antibodies. Antibodies against CDllb (clone ICRF44, BioLegend), CD86 (clone 2331, BioLegend), HLA- DR (clone L243, BioLegend), and CD80 (clone L307, BD) were used for marker staining. FVS786 viability staining was used to exclude dead cells from analysis. The mean fluorescence intensity of stained cells was measured by CytoFlex flow cytometry and analyzed by Kaluza software (Beckman Coulter).

Intracellular HIV virus detection- 2 long-terminal repeat (LTR)- DNA circles quantitation [0030] DNA of 3 day-cultured PBMC (3xl0 ⁶ cells/well in a 24-well culture plate) treated with/without PMA (lOOng/ml) and ionomycin (1 pg/ml) in the presence of human IgG4 antibody (BioLegend) or Anti-Mac-1 antibody (H4L2, 10 pg/ml) was extracted with QIAamp DNA Blood Mini Kit (Qiagen, MD, USA) and DNA were eluted by 50pl nuclease-free water. Digital PCR was performed with the QX100™ Droplet Digital™ PCR platform (Bio-Rad, Hercules, California). The ddPCR mix was made by adding 1-5 pl of sample to 10 pl 2x ddPCR™ supermix for probes (Bio-Rad), Ipl EcoR, 500 nM of primers, and 250 nM of probe in a final volume of 20 pl. The mix was placed in an 8-channel cartridge, 70 pl of droplet generating oil (Bio-Rad) was added and droplets were generated in the QX100™ droplet generator (Bio-Rad). Droplet in oil suspensions were transferred to an ddPCR 96-well plate (Bio-Rad) and PCR was performed in the T100™ Thermal Cycler (Bio-Rad). DdPCR amplification reactions consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec denaturation and 60°C for 60 sec annealing/elongation temperature, and enzyme deactivation at 98°C for 10 min. The ramping rate of each step is 2°C/sec. The sequences of primer pairs are listed in Table VI.

Statistical analysis

[0031] Results were compared by Fisher’s exact test for categorical variables and paired t test or unpaired t test for continuous variables as appropriate. Data are reported as the mean ± SEM. Statistical analysis was performed using Prism 9.0 software. Two-sided tests were used, and a p-value of < 0.05 was considered statistically significant.

Results

Expression of heterodimeric CDllb/CD18 (Mac-1) on HEK293 cell surface [0032] HEK293 cells, which do not express endogenous Mac-1, were transfected with pcDN A3.1 /human CDl lb and pcDN A3.1 /human CD18 plasmids using liposome transfections. After G418 and hygromycin selections, we obtained several single-cell clones stably expressing the human Mac-1 on the cell surface by FACS sorting using CD18-specific mAb (clone 6.7) and CDllb specific mAb (clone ICRF44). One clone, designated 1-4, was selected for all the examples presented in this description. Other clones show similar properties.

[0033] The expressions of CDllb and CD 18 on the HEK293 cells, as detected by flow cytometry, are shown in FIG 2. We verified the conformational state on the surface of HEK293/Mac-1 cells using two activation-sensitive antibodies, mAbs, KIM127 and m24. KIM127 can fully bind to HEK293/Mac-1, suggesting that Mac-1 is in the extended conformation. Little binding of m24 to HEK293/Mac-1 cells were observed in PBS (Mock), suggesting that Mac-1 is in the low affinity state. The HEK293/Mac-1 in the PBS is partially activated. In contrast, Mn ²⁺ treatment induced 100% of Mac- 1 molecule to adopt an extended, high-affinity conformation. Thus, these cells provide an excellent platform for the screening of monoclonal antibodies of human Mac-1.

Anti-Mac- 1 antibodies selectively bind to the different states of Mac- 1 on HEK293/Mac-l cell surface

[0034] We constructed human antibody and mouse antibody phage display libraries and then screened and isolated clones carrying specific antibody genes that can recognize Mac-1. A total of 79 clones were picked from the phage pools from 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 its native conformation, we generated recombinant antibodies from these clones with human IgG4 backbone. The recombinant anti -Mac- 1 antibodies were used in 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.

[0035] Conformational change of Mac-1 is involved in the regulation of its functions. We examine whether these antibodies can recognize different conformations of Mac-1. As listed in Table II, some clones selectively bind to an activation-specific epitope on Mac-1 molecules on HEK293/Mac-1 cells after stimulation with Mn ²⁺ (Mock/MnCh Ratio < 1). In contrast, some clones preferentially recognize the resting form of Mac-1 (Mock/MnCh Ratio > 1). The deduced amino-acid sequences of the CDRs and framework regions of selected clones are shown in Table I. Anti-Mac- 1 antibodies predominantly bind to Mac-1 on the innate immune cell surface [0036] The innate immune cells such as monocytes (CD14 ⁺ cells) and neutrophils (CD66b ⁺ cells) are the main cells that express Mac-1 on their cell surface. Some populations of B cells also expressed Mac-1 on their cell surface (Proc Natl Acad Sci U S A. 2008 Apr 1; 105(13):5195- 200). The specificities of selective anti-Mac-1 antibodies were determined by flow cytometry using human whole blood. As shown in FIG. 3A, anti-Mac-1 antibodies in this example were able to bind to the innate immune cells (CD14 ⁺ and CD66b ⁺ cells) and small populations of B cells (CD19 ⁺ cells). In contrast, these antibodies did not bind to T cells (CD3 ⁺ lymphocytes). Taken together, these results indicate that anti-Mac-1 antibodies can specifically bind to the Mac-1 epitope on the immune cells. To determine whether an anti-Mac-1 antibody validated for human Mac-1 will cross-react with the mouse Mac-1, we use Raw 264.7 mouse macrophage cell line that expressed mouse Mac-1 on the cell surface. As shown in FIG. 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.

Anti -Mac- 1 antibodies induce a conformational change in Mac-1

[0037] It is well known that inside-out signaling induces global conformational changes of Mac-1 leading to outside-in signaling. To screen which anti -Mac- 1 antibodies would induce conformational changes in Mac-1, we used KIM127 and m24 antibodies, which bind preferentially to Mac-1 in the extended conformation, as reporters to detect conformational changes. As shown in FIG. 4 left panel, incubation of the antibodies with HEK293/Mac-1 cells in PBS buffer (Mock) resulted in basal levels of KIMI 27 and m24 bindings. In contrast, incubation of the antibodies with HEK293/Mac-1 cells in MnCh/PBS buffer (Mn ²⁺ ) induced maximal levels of KIM127 and m24 bindings (FIG.4 right panel). Incubation with DF3M-5 in the Mock condition induced a small increase in KIM127 binding and a large increase in m24 binding (FIG. 4 left panel), indicating that this DF3M-5 clone can serve as an agonist to enhance the conformational change of Mac-1. Other clones that can serve as agonists (based on m24 expression relative IgG4 control >1) are listed in Table III. In contrast, incubation with 28E07 in the Mn ²⁺ condition induces a small decrease in KIM 127 binding and a large decrease in m24 binding (FIG. 4 right), indicating that this 28E07 clone can serve as an antagonist to reduce the conformational change of Mac-1. Other clones that can serve as antagonists (based on m24 expression relative IgG4 control <1) are listed in Table IV. Results from these studies indicate that antibodies of the invention may be selectively used to control the conformational changes of Mac- 1, thereby regulating the functions of Mac- 1. Anti-Mac-1 antibodies modulate Thl/Th2 cytokine secretion by TLR-activated immune cells in vivo,

[0038] Previous studies show that active CD 11b integrin engages in crosstalks with the MyD88 and TRIF pathways and modulate TLR signaling in innate immune responses (Nat Immunol. 2010 Aug; 11 (8): 734-42). To examine whether anti-Mac-1 antibodies of the invention can modulate Thl/Th2 cytokine secretions in TLR-activated immune cells in vivo, Balb/c mice (n=4/group) were intraperitoneal injected with 5 mg/kg LPS and 10 mg/kg anti-Mac-1 antibodies. Four hours later, serum Thl/Th2 cytokines were measured by ProcartaPlex™ MS. As shown in FIG 5, m2396 and DF3M-5 treatments enhanced TLR4-induced Thl cytokines (such as IFN-y, IL-1 , and TNF-a) and slightly enhance TLR4-induced Th2 cytokines (such as IL-5 and IL-13) in the serum. These results suggest that anti-Mac-1 antibody (clones m2396 and DF3M-5) treatment can skew the TLR-induced Thl/Th2 responses. In contrast, 24G05 treatment didn’t alter Thl/Th2 cytokine profile.

Anti-Mac- 1 antibody treatment reduces tumor growth

[0039] The anti-cancer activities of the anti-Mac-1 antibodies of the invention (e.g., m2396 and 28E07-HH) were further evaluated in the treatment of A549 cancer model in female NOG- EXL humanized mice.

[0040] When the average tumor volumes reached about 41 mm ³ , tumor bearing mice were randomized into 3 groups (Human IgG4, m2396, and 28E07-HH) and the treatments were started. The mean tumor sizes of mice reached 172.59 mm ³ in Human IgG4 group, 132.51 mm ³ in m2396 group, and 109.88 mm ³ in 28E07-HH group on Day35 post grouping (FIG. 6). FIG. 6 shows results from representative antibodies m2396 and 28E07-HH. Other antibodies of the invention have similar properties. The tumor growth inhibition (TGI) % of the m2396 group and 28E07-HH group were 23.59%, and 35.93%, respectively. The TGI of the different groups at different time points were shown in Table V. These results indicate that these anti -Mac- 1 antibodies can serve as therapeutic antibodies to treat human cancer.

Anti-Mac- 1 antibody treatment reduced HIV viral load and reverses immunosuppressed phenotype of PBMC in HIV patients

[0041] To test the efficacy of the anti-Mac-1 antibody-mediated inhibitory actions against HIV, PBMC isolated from fifteen latent HIV -infected patients were treated with anti -Mac- 1 antibodies for 3 days in vitro. As shown in FIG 7A, the anti-Mac-1 antibody (H4L2 shown as a representative of anti-Mac-1 antibodies) significantly enhanced the expression of CD86 and MHC class II functional markers in myeloid cells of HIV patients. These results indicate that enhanced T cell activation and dendritic cells (DCs) maturation in HIV patients can be achieved with the anti-Mac-1 antibodies of the present invention. These enhanced immune responses may suggest a potential application for the antibodies of the invention in the treatment of HIV infection.

[0042] While combination antiretroviral therapy (ART) may suppress HIV replication. HIV- 1 persists in the infected cells as a stable integrated genome and more labile unintegrated DNA, which includes linear, 1-LTR and 2-LTR circular DNA. 2-LTR circle DNA, although less abundant, is considered a surrogate marker for 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/sl2967- 020-02368-y).

[0043] To detect the load of intracellular HIV virus, HIV virus DNA reservoir was quantified using the 2 long-terminal repeat (LTR) DNA circles as the marker. Because these fifteen HIV-1 infected patients were receiving regular highly active antiretroviral therapy (ART) treatments, only 3 of the 15 patients had detectable levels of the HIV DNA by the LTR assay. Nevertheless, declines in the HIV 2LTR DNA levels were observed in these 3 patients’ PBMC samples treated with the anti -Mac- 1 antibody or with the anti-Mac-1 antibody in combination with phorbol 12- myristate 13-acetate (PMA) and ionomycin (FIG. 7B).

[0044] While the invention has been described with a limited number of examples, one skilled in the art would appreciate that these examples are for illustration only and that other modifications and variations are possible without departing from the scope of the invention. Therefore, the scope of protection should only be limited by the attached claims.

Tables

Table I: Heavy-chain variable region sequences and light-chain variable region sequences of SEQ ID NO:1 through SEQ ID NO:158

Anti-Mac- 1 antibody sequence clone: 24F08 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24F09 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24F11 (Underline is a CDR sequence) Anti-Mac- 1 antibody sequence clone: 24F12 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 24G01 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G05 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G07 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G08 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G09 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G10 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24G11 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 24G12 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 24H01 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24H02 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 24H03 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25A02 (Underline is a CDR sequence) Anti-Mac- 1 antibody sequence clone: 25A04 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25A06 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25A09 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25A10 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25B03 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25B04 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 25B01 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3-10 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3-28 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3-30 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3-32 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-16 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-17 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-22 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-25 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-26 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4-42 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF3M-1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF3M-2 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF3M-5 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3M-18 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF3M-19 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3M-30 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF3M-36 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF3M-42 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-3 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF4M-7-1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-7-4 (Underline is a CDR sequence) Anti-Mac- 1 antibody sequence clone: DF4M-9 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF4M-11 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-17 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF4M-18 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-21 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-23 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF4M-30 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: DF4M-31 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: DF4M-45 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 28A12 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 27G04 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 27A04 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 27A06 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28G06 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 27B10 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 27D06 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28D06 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 27E12 (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: m2396 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: H4L2 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-HH (Underline is a CDR sequence)

Anti-Mac-1 antibody sequence clone: 28E07-B1H (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B2H (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B3H (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B4H (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-HB1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-HB2 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-HB3 (Underline is a CDR sequence) Anti-Mac- 1 antibody sequence clone: 28E07-HB4 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B1B1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B1B2 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B2B1 (Underline is a CDR sequence)

Anti-Mac- 1 antibody sequence clone: 28E07-B2B2 (Underline is a CDR sequence)

Table II: The anti-Mac-1 antibodies selectively bind to different statuses of human Mac-1.

HEK293/Mac-1 cells (clonel-4) were incubated in PBS (Mock), or PBS/MnCh (Mn ²⁺ ).

Binding of isotype control IgG, the CDllb specific mAb (ICRF44), the CDllb activationsensing mAh (CBRM1/5), or the screened anti-Mac-1 antibody was detected using flow cytometry.

Table III: The anti-Mac-1 antibodies can serve as agonist to enhance the conformational change of Mac- 1. HEK293/Mac-1 cells (clonel-4) were incubated with 10 pg/ml anti-Mac-1 antibodies under the PBS (Mock) condition. Binding of KIM127 or m24 was detected using flow cytometry.

Table IV: The anti-Mac-1 antibody can serve as an antagonist to reduce the conformational change of Mac- 1. HEK293/Mac-1 cells (clonel-4) were incubated with 10 Hg/ml anti-Mac-1 antibodies under the PBS/MnCh (Mn ²⁺ ) condition. Binding of KIMI 27 or m24 was detected using flow cytometry.

Table V: Tumor growth inhibition (TGI, %)

Table VI: Primer and probe sequences used in digital PCR

Table VII: Heavy-chain CDR region sequences and light-chain CDR region sequences of

SEQ ID NO: 159 through SEQ ID NO: 554

Anti-Mac- 1 antibody CDR sequence clone: 24F08

Anti-Mac- 1 antibody CDR sequence clone: 24F09

Anti-Mac- 1 antibody CDR sequence clone: 24F11

Anti-Mac- 1 antibody CDR sequence clone: 24F12

Anti-Mac-1 antibody CDR sequence clone: 24G01

Anti-Mac- 1 antibody CDR sequence clone: 24G05

Anti-Mac- 1 antibody CDR sequence clone: 24G07

Anti-Mac- 1 antibody CDR sequence clone: 24G08 Anti-Mac- 1 antibody CDR sequence clone: 24G09

Anti-Mac- 1 antibody CDR sequence clone: 24G10

Anti-Mac-1 antibody CDR sequence clone: 24G11

Anti-Mac-1 antibody CDR sequence clone: 24G12

Anti-Mac-1 antibody CDR sequence clone: 24H01

Anti-Mac- 1 antibody CDR sequence clone: 24H02

Anti-Mac- 1 antibody sequence clone: 24H03 (Underline is a CDR sequence)

Anti-Mac- 1 antibody CDR sequence clone: 25A02

Anti-Mac- 1 antibody CDR sequence clone: 25A04

Anti-Mac- 1 antibody CDR sequence clone: 25A06

Anti-Mac- 1 antibody CDR sequence clone: 25A09 Anti-Mac- 1 antibody CDR sequence clone: 25A10

Anti-Mac- 1 antibody sequence clone: 25B03 (Underline is a CDR sequence)

Anti-Mac- 1 antibody CDR sequence clone: 25B04

Anti-Mac- 1 antibody CDR sequence clone: 25B01

Anti-Mac-1 antibody CDR sequence clone: DF3-10

Anti-Mac-1 antibody CDR sequence clone: DF3-28

Anti-Mac-1 antibody CDR sequence clone: DF3-30

Anti-Mac-1 antibody CDR sequence clone: DF3-32

Anti-Mac-1 antibody CDR sequence clone: DF4-16

Anti-Mac-1 antibody CDR sequence clone: DF4-17

Anti-Mac- 1 antibody CDR sequence clone: DF4-22 Anti-Mac- 1 antibody CDR sequence clone: DF4-25

Anti-Mac- 1 antibody CDR sequence clone: DF4-26

Anti-Mac- 1 antibody CDR sequence clone: DF4-42

Anti-Mac- 1 antibody CDR sequence clone: DF3M-1

Anti-Mac- 1 antibody CDR sequence clone: DF3M-2

Anti-Mac- 1 antibody sequence clone: DF3M-5 (Underline is a CDR sequence)

Anti-Mac-1 antibody CDR sequence clone: DF3M-18

Anti-Mac- 1 antibody CDR sequence clone: DF3M-19

Anti-Mac-1 antibody CDR sequence clone: DF3M-30

Anti-Mac-1 antibody CDR sequence clone: DF3M-36

Anti-Mac- 1 antibody CDR sequence clone: DF3M-42 Anti-Mac- 1 antibody CDR sequence clone: DF4M-1

Anti-Mac- 1 antibody CDR sequence clone: DF4M-3

Anti-Mac- 1 antibody CDR sequence clone: DF4M-7-1

Anti-Mac- 1 antibody CDR sequence clone: DF4M-7-4

Anti-Mac- 1 antibody CDR sequence clone: DF4M-9

Anti-Mac-1 antibody CDR sequence clone: DF4M-11

Anti-Mac- 1 antibody CDR sequence clone: DF4M-17

Anti-Mac-1 antibody CDR sequence clone: DF4M-18

Anti-Mac- 1 antibody CDR sequence clone: DF4M-21

Anti-Mac- 1 antibody CDR sequence clone: DF4M-23

Anti-Mac-1 antibody CDR sequence clone: DF4M-30 Anti-Mac-1 antibody CDR sequence clone: DF4M-31

Anti-Mac- 1 antibody CDR sequence clone: DF4M-45

Anti-Mac-1 antibody CDR sequence clone: 28E07, 28E07-HH, 28E07-B1H, 28E07-B2H, 28E07-B3H, 28E07-B4H, 28E07-HB1, 28E07-HB2, 28E07-HB3, 28E07-HB4, 28E07-B1B1, 28E07-B1B2, 28E07-B2B1, and 28E07-B2B2

Anti-Mac-1 antibody CDR sequence clone: 28A12

Anti-Mac-1 antibody CDR sequence clone: 27G04

Anti-Mac- 1 antibody CDR sequence clone: 27A04

Anti-Mac- 1 antibody CDR sequence clone: 27A06

Anti-Mac- 1 antibody CDR sequence clone: 28G06

Anti-Mac- 1 antibody CDR sequence clone: 27B10

Anti-Mac- 1 antibody CDR sequence clone: 27D06

Anti-Mac- 1 antibody CDR sequence clone: 28D06

Anti-Mac-1 antibody CDR sequence clone: 27E12

Anti-Mac-1 antibody CDR sequence clone: m2396

Anti-Mac- 1 antibody CDR sequence clone: H4L2