EA043281B1 - ANTI-CD47 ANTIBODIES AND THEIR USE - Google Patents

Description

Cross-references to related applications

This application claims priority over a U.S. provisional application

62/540118, filed August 2, 2017, and U.S. Provisional Application 62/657,094, filed April 13, 2018, each of which is incorporated herein in its entirety by reference.

The field of technology to which the invention belongs

The present invention relates to monoclonal anti-CD47 antibodies, nucleic acids and expression vectors encoding these antibodies, recombinant cells containing these vectors, and compositions containing these antibodies. Also provided are methods for producing antibodies and methods for using these antibodies to treat diseases, including cancer, inflammatory diseases, infectious diseases, arteriosclerosis, cardiovascular disease, metabolic disorders, radiation injury, and/or autoimmune diseases.

Link to the sequence listing provided in the electronic version

The present application contains a sequence listing, which is submitted electronically via EFS-Web as a sequence listing in ASCII format with the file name 689204.2WO Sequence Listing and the file creation date July 05, 2018 with a size of 95 kB. The sequence listing provided via EFS-Web is part of the description and is incorporated herein by reference in its entirety.

State of the art

Malignant cells can develop various protective abilities to avoid attack from the host, including from the immune system. They either give their cell surface a natural appearance, corresponding to the cell surface of a normal human cell, or interrupt the immune attack when taken over by immune cells. The latter mechanism is unequivocally supported by unexpected successes obtained with therapeutic monoclonal antibodies targeting the immunosuppressors CTLA-4, PD-1, and PD-L1. These antibodies inactivate immune checkpoints and allow T cells to effectively attack cancer cells, thus providing long-term efficacy in some patients. This recent success driven by these antibodies has rejuvenated the field of immuno-oncology and inspired research and development of more therapeutics to mobilize the human immune system to fight cancer.

The human immune system consists of adaptive (acquired) and innate immunity. Modern checkpoint blockers and modulators of the tumor microenvironment, used in clinical practice or in pharmaceutical development, are aimed at adaptive immunity. Checkpoint blockers and modulators of the tumor microenvironment mobilize T cells, saving T-helpers and T-killers from depletion, depleting immunosuppressive regulatory T cells, or blocking the formation of an immunosuppressive tumor microenvironment. More recently, new evidence has emerged indicating that tumor cells also suppress innate immunity, and attenuation of this suppression has shown great therapeutic potential in vitro and in vivo in the treatment of malignant tumors.

Innate immunity is the first line of defense against invading pathogens. It consists of defense mechanisms and leukocytes that absorb antigens. Among them are macrophages, which remove non-functional old and infected host cells by phagocytosis. Tumor cells also avoid macrophage attack by overexpressing cluster of differentiation 47 (CD47) (also known as integrin-associated protein), a marker that is also ubiquitously expressed on the surface of normal cells. Notably, in the presence of antibodies that specifically block CD47, macrophages attack tumor cells in vitro in a phagocytosis assay and kill tumors in vivo in xenograft models. Currently, several therapeutic agents targeting CD47 have entered the phase of clinical drug development.

CD47, first identified as an integrin-related protein, is a receptor ligand and interacts with many proteins. CD47 is the receptor for thrombospondin-1 (TSP1), one of the best characterized secreted ligands. On the other hand, CD47 is a ligand involved in the signaling of regulatory protein alpha (SIRPa), an inhibitory receptor expressed on the surface of macrophages. It is the binding of the latter that prevents macrophages from entering cancer cells.

As with all cancer immunotherapies, CD47 blocking can induce an inadvertent immune attack on normal cells resulting in dose-limiting toxicity. Indeed, the CD47-blocking antibody B6H12 causes hemagglutination, presumably by binding to CD47 on erythrocytes. Notably, this antibody blocks the binding of TSP1 and SIRPa to CD47. However, it remains unclear whether blocking the interaction of TSP1 with B6H12 is the cause of hemagglutination. In addition, it is hypothesized that macrophages can attack normal cells when their surface CD47 molecules are masked by systemically administered anti-CD47 antibodies. Therefore, it is very important to develop anti-CD47 antibodies that have increased specificity.

- 1 043281 to tumor cells and reduced toxicity to normal cells.

The essence of the invention

In one general aspect, the invention relates to isolated monoclonal antibodies, or antigen-binding fragments thereof, that bind to CD47.

Isolated monoclonal antibodies or their antigen-binding fragments are proposed, containing complementarity determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3, complementarity determining region 1 of the light chain (LCDR1), LCDR2 and LCDR3, having polypeptide sequences:

(1) SEQ ID NOs: 177, 46, 47, 178, 112 and 179, respectively;

(2) SEQ ID NOs: 51, 52, 53, 117, 118 and 119, respectively;

(3) SEQ ID NOs: 54, 55, 56, 120, 121 and 122, respectively;

(4) SEQ ID NOs: 57, 58, 59, 123, 124 and 125, respectively;

(5) SEQ ID NOs: 60, 61, 62, 126, 127 and 128, respectively;

(6) SEQ ID NOs: 180, 181, 182, 129, 130 and 131, respectively;

(7) SEQ ID NOs: 72, 73, 74, 138, 139 and 140, respectively;

(8) SEQ ID NOs: 78, 79, 80, 144, 145 and 146, respectively;

(9) SEQ ID NOs: 81, 82, 83, 147, 148 and 149, respectively;

(10) SEQ ID NOs: 84, 85, 86, 150, 151 and 152, respectively;

(11) SEQ ID NOs: 87, 88, 89, 153, 154 and 155, respectively;

(12) SEQ ID NOs: 90, 91, 92, 156, 157 and 158, respectively;

(13) SEQ ID NOs: 93, 94, 95, 159, 160 and 161, respectively;

(14) SEQ ID NOs: 96, 97, 98, 162, 163 and 164, respectively;

(15) SEQ ID NOs: 99, 100, 101, 165, 166 and 167, respectively;

(16) SEQ ID NOs: 102, 103, 104, 168, 169 and 170, respectively;

(17) SEQ ID NOs: 105, 106, 107, 171, 172 and 173, respectively;

(18) SEQ ID NOs: 108, 109, 110, 174, 175 and 176, respectively; or (19) SEQ ID NOs: 201, 202, 203, 204, 205 and 206, respectively;

wherein the antibody, or antigen-binding fragment thereof, specifically binds to CD47, preferably human CD47. SEQ ID NO: 177 is represented by the amino acid sequence GYTFTX1YY where X1 is an amino acid selected from D or A. SEQ ID NO: 178 is represented by the amino acid sequence X1NVGTY where X1 is an amino acid selected from D or E. SEQ ID NO: 179 is the amino acid sequence GQX1YSYPLT, where X1 is an amino acid selected from S or T. SEQ ID NO: 180 is represented by the amino acid sequence GYTFTSX1W, where X ₁ is an amino acid selected from S or Y. SEQ ID NO: 181 is represented by the amino acid sequence IDPSDSEX1, where X1 is an amino acid selected from T or A. SEQ ID NO: 182 is represented by the amino acid sequence X1RWGYYGKSAX2DY, where X1 is an amino acid selected from A or S and X ₂ is an amino acid selected from I or M.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment comprises a heavy chain variable region with a polypeptide sequence that is at least 95% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 , 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43, or a light chain variable region with a polypeptide sequence that is at least 95% identical to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment contains:

(a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 2;

(b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 4;

(c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 6;

(d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 8;

(e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 10;

(f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 12;

(g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 14;

(h) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 16;

- 2 043281 (i) a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 17 and a light chain variable region with a polypeptide sequence of SEQ ID NO: 18;

(j) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 19 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 20;

(k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 22;

(l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 23 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 24;

(m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 25 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 26;

(n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 27 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 28;

(o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 30;

(p) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 31 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 32;

(q) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 33 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 34;

(r) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 35 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 36;

(s) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 37 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 38;

(t) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 39 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 40;

(u) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 41 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 42; or (v) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 44.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, is chimeric.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, is human or humanized. In some embodiments, the humanized monoclonal antibody, or antigen-binding fragment thereof, comprises:

a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

h) the heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and the light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

i) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

j) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 198;

k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 187 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 196;

n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 197; or

o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 199 and

- 3 043281 light chain variable region with the polypeptide sequence of SEQ ID NO: 200.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, is capable of blocking CD47 binding to thrombospondin-1 (TSP1) and/or signal regulatory protein alpha (SIRPa).

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is capable of inducing macrophage-mediated phagocytosis of cancer cells.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, is capable of binding to cancer cells with minimal to undetectable binding to erythrocytes.

Also provided are isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention.

Also provided are vectors containing isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof according to the invention.

Also provided are host cells containing vectors containing isolated nucleic acids encoding monoclonal antibodies or antigen-binding fragments thereof according to the invention.

In some embodiments, a pharmaceutical composition is provided comprising said isolated monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

Also provided are methods for blocking the binding of CD47 to thrombospondin-1 (TSP1) and/or CD47 to signal regulatory protein alpha (SIRPa) in a patient, comprising administering to an individual the pharmaceutical compositions of the invention.

Also provided are methods of treating cancer in a patient, comprising administering to an individual the pharmaceutical compositions of the invention. The malignant tumor may be any type of hemoblastosis or solid cancer, for example, it may be selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, cancer breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblast leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other types of hemoblastosis.

Also provided are methods of treating an inflammatory disease in a patient, comprising administering to an individual the pharmaceutical compositions of the invention.

Also provided are methods of treating an infectious disease in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

Also provided are methods for treating arteriosclerosis in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

Also provided are methods of treating cardiovascular disease in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

Also provided are methods for treating a metabolic disorder in a patient, comprising administering to an individual the pharmaceutical compositions of the invention.

Also provided are methods for treating radiation injury in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

Also provided are methods of treating an autoimmune disease in a patient, comprising administering to an individual the pharmaceutical compositions of the invention.

Methods for determining the level of CD47 in an individual are also provided. The methods include (a) obtaining a sample from an individual; (b) bringing the sample into contact with the antibody or antigen-binding fragment of the invention; and (c) determining the level of CD47 in the individual. In some embodiments, the sample is a tissue or blood sample. The tissue sample may be, for example, a sample of cancerous tissue. The blood sample may contain, for example, cancer cells.

Methods for producing a monoclonal antibody or antigen-binding fragment thereof according to the invention are also provided, including culturing a cell containing a nucleic acid encoding a monoclonal antibody or antigen-binding fragment under conditions for producing a monoclonal antibody or antigen-binding fragment, and isolating the antibody or antigen-binding fragment from the cell or culture.

Also provided are methods for preparing a pharmaceutical composition containing a monoclonal antibody or antigen-binding fragment of the invention, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

- 4 043281

Brief description of the drawings

The above summary of the invention, as well as the following detailed description of the preferred embodiments of the present invention will be better understood when studied with reference to the accompanying drawings. However, it should be understood that the application is not limited to the specific embodiments shown in the drawings.

Fig. 1 is a plot of anti-CD47 mAb binding to RAJI cells according to FACS analysis. Control groups of CTL Mu 2° Ab and CTL Hu 2° Ab were treated not with primary antibodies, but with AlexaFluor 488 conjugated anti-mouse and anti-human secondary Ab IgG, respectively.

In FIG. 2A-2P are plots of anti-CD47 mAb activity blocking the interaction between CD47(ECD)-HIS and SIRPa-huFc as analyzed by ELISA. Curves were generated and IC5 ₀ values were calculated using Prism GraphPad version 7.

In FIG. 2A is a plot of anti-CD47 mAb 15G23A activity.

In FIG. 2B is a graph of anti-CD47 mAb 17C6A activity.

In FIG. 2C is a plot of anti-CD47 mAb 13B18A activity.

In FIG. 2D is a plot of anti-CD47 mAb 4M8A activity.

In FIG. 2E is a graph of anti-CD47 mAb 14D18A activity.

In FIG. 2F is a graph of anti-CD47 mAb 11G2A activity.

In FIG. 2G is a graph of anti-CD47 mAb 13C4A activity.

In FIG. 2H is a plot of anti-CD47 mAb 5D24A activity.

In FIG. 21 is a graph of the activity of the anti-CD47 mAb 9O23A.

In FIG. 2J is a plot of anti-CD47 mAb 17N8A activity.

In FIG. 2K is a plot of anti-CD47 mAb 14P6A activity.

In FIG. 2L shows a plot of anti-CD47 mAb 19L14A activity.

In FIG. 2M is a graph of anti-CD47 mAb 14O18A activity.

In FIG. 2N is a plot of anti-CD47 mAb 1J7A activity.

In FIG. 2O is a graph of anti-CD47 mAb 16M17A activity.

In FIG. 2P is a graph of the activity of anti-CD47 mAb 18M19A (a chimeric antibody with human IgG4 heavy chain and kappa light chain).

In FIG. 3A-3B show the evaluation of anti-CD47 mAb in a haemagglutination assay using fresh donated blood. Purification buffer, B6H12, and PBS were used as controls. Antibody concentrations are indicated above the panel. In FIG. 3A shows the results of the hemagglutination assay for anti-CD47 mAb 14P6A, 11F6A, 18M19A, 19L14A, 305A, 10I23A, 14N13A, 14O18A, 13C4A, 16M17A, and 17O12A. In FIG. 3B shows the results of the hemagglutination assay for anti-CD47 mAb 12B18A, 4M8A, 13B18A, 11G2A, 5D24A, 14D18A, 17C6A, 17N8A, 9O23A, 15G23A and 1J7A and PBS and B6H12 controls.

In FIG. 4A-4C are plots of in vivo antitumor activity, body weight, and serum concentration in mice treated with anti-CD47 mAb 13B18A-huIgG1.

In FIG. 4A shows the in vivo antitumor activity of 13B18A-huIgG1 in a mouse RAJI xenograft model; Rituximab was used as a positive control.

In FIG. 4B shows the body weight data of animals in different groups obtained during the study.

In FIG. 4C shows the serum concentration of 13B18A-huIgG1 in the mAb treated groups 2 days after the last dose.

In FIG. 5A and 5B show the activity of the humanized anti-CD47 mAb H3L9 (FIG. 5A) and H5L5 (FIG. 5B) blocking the interaction between human CD47(ECD)-HIS and huSIRPa-muFc as analyzed by ELISA.

Fig. 6 shows the results of a haemagglutination assay with humanized mAbs H3L9, H5L5 and H8L10. Mouse mAb 15G23A was used as a positive control.

Fig. 7A-7B show the results of a binding assay of erythrocytes (RBCs) (FIG. 7A) and RAJI cells (FIG. 7B) with humanized H3L9, H5L5, and H8L10 mAbs.

In FIG. 8 shows the activity of humanized anti-CD47 mAbs H3L9, H5L5 and H8L10 to block huSIRPa-muFc binding to RAJI cells. Only the 2nd Ab and no mAb/no control 2nd Ab are negative controls.

In FIG. 9 shows the activity of humanized anti-CD47 mAb H3L9, H5L5 and H8L10 on macrophage-induced phagocytosis of RAJI cells.

- 5 043281

Detailed description of the invention

In the Background section and in the description, references are made to or various publications, articles, and patents are described; each of these documents is incorporated herein in its entirety by reference. Discussion of documents, acts, materials, devices, articles, or the like, is included in the present description to disclose the context of the present invention. Such discussion is not an admission that each of the matters discussed are part of the prior art relating to any disclosed or claimed invention.

Unless otherwise indicated, all technical and scientific terms used in this specification have the same meanings as they are commonly understood by those skilled in the art to which the present invention pertains. Otherwise, certain terms used in this application have the meanings specified in the description.

It should be noted that the singular forms used in the present description and the appended claims include the plural, unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values, such as concentration or range of concentrations, described in this application, in all cases should be considered as modified by the term approximately. Thus, the numerical value usually includes ±10% of the specified value. For example, a concentration of 1 mg/ml includes 0.9 to 1.1 mg/ml. Similarly, the concentration range from 1 to 10% (w/v) includes from 0.9 to 11% (w/v). As used herein, use of a numeric range explicitly includes all possible subranges, all individual numeric values within that range, including integers and fractional values within such ranges, unless the context explicitly dictates otherwise.

Unless otherwise indicated, the term at least preceding a series of elements is to be understood as referring to each element of the series. Those skilled in the art will recognize, or be able to determine by routine routine experimentation, many embodiments of the invention equivalent to those disclosed herein. Such equivalents are within the scope of the present invention.

As used herein, the terms contains, containing, includes, including, has, having, comprising or comprising, or any variation thereof, means the inclusion of the specified integer or group of integers, and not the exclusion of any other integer or groups of integers, and should be understood as non-exclusive or open. For example, a composition, mixture, process, method, article, or device that contains a list of elements is not necessarily limited to only those elements, but may include other elements not explicitly listed or inherent in such composition, mixture, process, method, article, or device. Also, unless explicitly stated otherwise, the conjunction or refers to an inclusive or, not an exclusive or. For example, condition A or B is satisfied by any of the following conditions: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are correct (or present).

Used in the present description, the term and/or between several listed elements should be understood as covering both separate and combined options. For example, when two elements are connected by the union and/or, the first option refers to the possibility of using the first element without the second. The second option refers to the possibility of using the second element without the first. The third option refers to the possibility of using the first and second elements together. All of these options are included in the scope of the specified value and, therefore, satisfy the requirement of the term and/or used in the present description. It is clear that the possibility of using more than one of these options at the same time corresponds to the specified meaning and, therefore, satisfies the requirement of the term and/or.

In the context of the present invention, the term consists of, or variations thereof, such as consist of or consisting of, as used in the specification and claims, indicates the inclusion of any recited integer or group of integers, but no additional integer or group of integers. may be added to the specified method, structure, or composition.

In the context of the present invention, the term essentially consists of, or variations thereof, such as essentially consist of or essentially consists of, as used in the specification and claims, indicates the inclusion of any reducible integer or group of integers, and the optional inclusion of any reducible integer numbers or groups of integers that do not significantly change the basic or new properties of the specified method, structure or composition. See M.P.E.P. §2111.03.

In the context of the present description, the term individual means any animal, preferably a mammal, most preferably a human. As used herein, the term mammal includes any mammal. Examples of mammals include, without limitation, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

The words right, left, below and above indicate directions in the drawings to which reference is made.

It should also be understood that the terms about, approximately, generally, substantially, and similar terms used herein when referring to a size or feature of a component of a preferred embodiment of the invention indicate that the described size/feature is not a strict boundary or setting, and does not exclude minor deviations from the specified values, which are functionally the same or similar, which is understandable to a person skilled in the art. At a minimum, such references that include a numerical parameter include a range of values that, in terms of mathematical and manufacturing principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), does not will change the least significant digit.

The terms identical or percent identity, in the context of two or more nucleic acids or polypeptide sequences (e.g., anti-CD47 antibodies, CD47 polypeptides, and the polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a certain percentage of amino acids. residues or nucleotides that are the same when compared and aligned to determine the maximum match, measured using one of the following sequence comparison algorithms or by visual inspection.

For sequence comparisons, one sequence is typically used as a reference sequence against which test sequences are compared. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the coordinates of the subsequence are indicated, and program parameters are assigned for the sequence comparison algorithm. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences relative to the reference sequence based on the program parameters.

Optimal alignment of sequences for comparison can be performed, for example, using the Smith-Waterman local homology algorithm (Smith & Waterman, Adv. Appl. Math. 2:482 (1981)), the Needleman-Wunsch pairwise alignment algorithm (Needleman & Wunsch, J Mol. Biol. 48:443 (1970)), by the Lipman-Pearson similarity search method, (Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988)), by implementing these algorithms in the form computer programs (GAP, BESTFIT, FASTA, and TFASTA at Wisconsin Genetics Software, Genetics Computer Group, 575 Science Dr., Madison, WI) or by visual assessment (see, for example, Current Protocols in Molecular Biology, F.M. Ausubel et al. , eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms suitable for determining percent identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. BLAST analysis software is available through the National Center for Biotechnology Information. This algorithm involves first identifying high score sequence pairs (HSPs) by identifying short words of length W in the requested sequence that either match or satisfy some threshold positive score T when aligned with a word of the same length in the database sequence. T is called the score threshold for words in a particular neighborhood (Altschul et al., supra). Said initial neighbor word matches in the neighborhood act as a starting point for initiating a search to find containing HSPs. Word matching continues in both directions along each sequence as long as the total alignment score can be increased.

The total score for nucleotide sequences is calculated using the parameters M (reward points for a pair of matching residues; always >0) and N (penalty points for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the total score. The continuation of the search for a word match in each direction is terminated when: the total number of alignment points is reduced by the number X compared to its maximum value reached; the total score drops to zero or below as a result of the summation of one or more negative-scoring residual alignments; or the end of some sequence is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLAST program (for nucleotide sequences) defaults to a word length (W) of 11, an expectation (E) of -10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, BLAST defaults to a word length (W) of 3, an expectation (E) of -10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989 )).

In addition to calculating the percent sequence identity, the BLAST algorithm also

- 7 043281 performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which is a measure of the probability that a match between two nucleotide or amino acid sequences can occur by chance. For example, a nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability when comparing a test nucleic acid sequence to a second reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An additional indication that the two nucleic acid or polypeptide sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is usually substantially identical to the second polypeptide, for example, if the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are essentially identical is that the two molecules hybridize to each other under stringent conditions.

As used herein, the terms inhibit, inhibitory, and inhibition mean a reduction in an activity, response, condition, disease, or other biological parameter. These terms may include, without limitation, complete suppression of an activity, response, condition, or disease. They may also include, for example, a 10% reduction in activity, response, condition, or disease compared to native or control levels. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any value in between as compared to native or control levels. As a non-limiting example, an antibody of the invention can inhibit the activity of the CD47 protein. The activity of the CD47 protein may be reduced or reduced relative to the native activity of the CD47 protein.

Antibodies.

The invention generally relates to isolated anti-CD47 antibodies, nucleic acids and expression vectors encoding these antibodies, recombinant cells containing the vectors, and compositions containing said antibodies. Also provided are methods for producing antibodies and methods for using these antibodies to treat diseases, including cancer, inflammatory diseases, autoimmune diseases, arteriosclerosis, cardiovascular disease, metabolic disorders, radiation injury, and/or infectious diseases. Antibodies of the invention have one or more of the desired functional properties, including, but not limited to, high affinity binding to CD47, high specificity for CD47, the ability and/or inability to block CD47 binding to thrombospondin-1 (TSP1), the ability to block CD47 binding to signal alpha regulatory protein (SIRPa), the ability to induce phagocytosis of CD47-expressing cells associated with a disease or disorder (including, but not limited to, cancer and arteriosclerosis), and the ability to inhibit tumor growth in animal models and in individuals when administered alone or in combination with other types of anticancer therapy and the inability to induce hemagglutination.

In a general aspect, the present invention provides isolated monoclonal antibodies, or antigen-binding fragments thereof, that bind to CD47.

In the context of the present description, the term antibody is used in a broad sense and includes immunoglobulin molecules or antibodies, including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. Typically, antibodies are proteins or peptide chains that exhibit binding specificity for a particular antigen. The structures of antibodies are well known. Immunoglobulins can be classified into five major classes (ie, IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into IGA1, IgA2, IgG1, IgG2, IgG3 and IgG4 isotypes. Accordingly, the antibodies of the invention may be any of the five major classes or respective subclasses. Preferably, an antibody of the invention is an IgG1, IgG2, IgG3 or IgG4. Light chain antibodies of vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, antibodies of the invention may comprise a kappa or lambda light chain constant domain. In accordance with specific embodiments, the antibodies of the invention comprise heavy and/or light chain constant regions derived from rat or human antibodies. In addition to heavy and light constant domains, antibodies contain an antigen-binding region that consists of a light chain variable region and a heavy chain variable region, each containing three domains (i.e., complementarity-determining regions 1-3; CDR1, CDR2, and CDR3 ). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2 and LCRD3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCRD2 and HCDR3.

- 8 043281

As used herein, an isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to CD47 is substantially free of antibodies that do not bind to CD47). In addition, the isolated antibody is substantially free of other cellular materials and/or chemicals.

In the context of the present description, the term monoclonal antibody refers to an antibody derived from a population of essentially homogeneous antibodies, i. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies of the invention can be produced by hybridoma, phage display, single lymphocyte gene cloning, or recombinant DNA techniques. For example, monoclonal antibodies can be produced using a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse or rat, having a genome containing a human heavy chain transgene and a light chain transgene.

In the context of the present description, the term antigen-binding fragment refers to an antibody fragment, such as, for example, diabody, Fab fragment, Fab', F(ab') ₂ , Fv, Fv fragment, stabilized by disulfide bonds (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized di-antibody (ds-antibody), single-chain antibody molecule (scFv), single-domain antibody (sdab), scFv-dimer (bivalent diabody), polyspecific antibody formed from a portion of an antibody, containing one or more CDRs, a camelid single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not contain the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or parental antibody fragment binds. In specific embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and a heavy chain Fd segment. In other specific embodiments, the antigen-binding fragment comprises Fab and F(ab').

As used herein, the term single chain antibody refers to a single chain antibody conventional in the art that includes a heavy chain variable region and a light chain variable region joined by a short peptide of about 15 to about 20 amino acids in length. As used herein, the term single domain antibody refers to a single domain antibody conventional in the art that contains a heavy chain variable region and a heavy chain constant region, or that contains only a heavy chain variable region.

In the context of the present description, the term human antibody refers to an antibody produced by a human body, or an antibody having an amino acid sequence corresponding to an antibody produced by a human body, obtained using any method known in this field. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies containing at least one human heavy and/or light chain polypeptide.

As used herein, the term humanized antibody refers to a non-human antibody that has been modified to increase sequence homology with a human antibody sequence while retaining the antigen-binding properties of the antibody, but with reduced antigenicity in humans.

As used herein, the term chimeric antibody refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of an antibody derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and ability, while the constant regions correspond to antibody sequences derived from another mammalian species (eg, human) to avoid initiating an immune response in that species.

In the context of the present description, the term polyspecific antibody refers to an antibody that contains a plurality of immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence of this set has binding specificity for the first epitope, and the second immunoglobulin variable domain sequence of this set has binding specificity for towards the second epitope. In one embodiment, the first and second epitopes are on the same antigen, such as the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or do not substantially overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the polyspecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

In the context of the present description, the term bispecific antibody refers to a polyspecific antibody that binds to no more than two epitopes or two antigens. A bespecifically antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for second epitope. In one embodiment, the bispecific antibody comprises a half-antibody or fragment thereof having binding specificity for a first epitope and a half-antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody comprises an scFv or fragment thereof having a binding specificity for a first epitope and an scFv or fragment thereof having a binding specificity for a second epitope. In one embodiment, the first epitope is located on CD47 and the second epitope is located on PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, apelin, DLL3, claudin 18.2, TIP-1, folate receptor alpha, CD3 and/or other tumor-associated immunosuppressants or surface antigens.

As used herein, the term CD47 refers to a transmembrane receptor belonging to the immunoglobulin superfamily, which has been shown to be involved in numerous cellular processes, including cell migration, adhesion, and T cell function. CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen (OA3), Rh-related antigen, and MER6, was initially identified as a tumor antigen in human ovarian cancer and has subsequently been shown to be expressed in many human tumors, including both hematologic and solid tumors. Interaction between CD47 and signal regulatory protein alpha (SIRPa), an inhibitory protein expressed on macrophages, prevents phagocytosis of cells expressing CD47. Low levels of CD47 are also expressed in virtually all non-malignant cells. The term human CD47 refers to human-derived CD47. An exemplary amino acid sequence of human CD47 is provided in GenBank under the number NP_001768.1 (SEQ ID NO: 207).

As used herein, an antibody that specifically binds to CD47 refers to an antibody that binds to CD47, preferably human CD47, with a KD of ^1x10'7 M or less, preferably 1x10'8 M or less, more preferably 5x10'9 M or less, 1x10'9 M or less, 5x10' ¹⁰ M or less, or 1x10' ¹⁰ M or less. The term KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (ie Kd/Ka) and expressed as a molar concentration (M). Antibody KD values can be determined using methods known in the art in light of the present disclosure. For example, the KD of an antibody can be determined using surface plasmon resonance, for example, using a biosensor system, such as the Biacore® system, or using biolayer interferometry technology, for example, using the Octet RED96 system.

The lower the KD value of an antibody, the higher the binding affinity of the antibody to the target antigen.

According to a specific aspect, the invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, a light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 having the polypeptide sequences:

(1) SEQ ID NO (2) SEQ ID NO (3) SEQ ID NO (4) SEQ ID NO (5) SEQ ID NO (6) SEQ ID NO (7) SEQ ID NO

177, 46, 47, 178, 112 and 179, respectively;

51, 52, 53, 117, 118 and 119, respectively;

54, 55, 56, 120, 121 and 122, respectively;

57, 58, 59, 123, 124 and 125, respectively;

60, 61, 62, 126, 127 and 128, respectively;

180, 181, 182, 129, 130 and 131, respectively;

72, 73, 74, 138, 139 and 140, respectively;

- 10 043281 (8) SEQ ID NOs: 78, 79, 80, 144, 145 and 146, respectively;

(9) SEQ ID NOs: 81, 82, 83, 147, 148 and 149, respectively;

(10) SEQ ID NOs: 84, 85, 86, 150, 151 and 152, respectively;

(11) SEQ ID NOs: 87, 88, 89, 153, 154 and 155, respectively;

(12) SEQ ID NOs: 90, 91, 92, 156, 157 and 158, respectively;

(13) SEQ ID NOs: 93, 94, 95, 159, 160 and 161, respectively;

(14) SEQ ID NOs: 96, 97, 98, 162, 163 and 164, respectively;

(15) SEQ ID NOs: 99, 100, 101, 165, 166 and 167, respectively;

(16) SEQ ID NOs: 102, 103, 104, 168, 169 and 170, respectively;

(17) SEQ ID NOs: 105, 106, 107, 171, 172 and 173, respectively;

(18) SEQ ID NOs: 108, 109, 110, 174, 175 and 176, respectively; or (19) SEQ ID NOs: 201, 202, 203, 204, 205 and 206, respectively;

wherein the antibody, or antigen-binding fragment thereof, specifically binds to CD47, preferably human CD47.

SEQ ID NO: 177 is represented by the amino acid sequence GYTFTX1YY, where X1 is an amino acid selected from D or A.

SEQ ID NO: 178 is represented by the amino acid sequence X1NVGTY, where X1 is an amino acid selected from D or E.

SEQ ID NO: 179 is represented by the amino acid sequence GQX1YSYPLT, where X1 is an amino acid selected from S or T.

SEQ ID NO: 180 is represented by the amino acid sequence GYTFTSX1W, where X1 is an amino acid selected from S or Y.

SEQ ID NO: 181 is represented by the amino acid sequence IDPSDSEX1, where X ₁ is an amino acid selected from T or A.

SEQ ID NO: 182 is represented by the amino acid sequence X1RWGYYGKSAX ₂ DY, where X1 is an amino acid selected from A or S and X2 is an amino acid selected from I or M.

In another specific aspect, the invention relates to an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43, or a light chain variable region with a polypeptide sequence , at least 85%, preferably 90%, more preferably 95% identical to one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 , 28, 30, 32, 34, 36, 38, 40, 42, or 44. In one preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, or 43, and a light chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 , 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44, respectively.

According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment according to the invention, containing:

a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 2;

b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 4;

c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 6;

d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 8;

e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 10;

f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 12;

g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 14;

h) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 16;

i) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 18;

j) a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 19 and

- 11 043281 ribable region of the light chain with the polypeptide sequence of SEQ ID NO: 20;

k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 22;

l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 23 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 24;

m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 25 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 26;

n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 27 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 28;

o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 30;

p) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 31 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 32;

q) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 33 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 34;

r) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 35 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 36;

s) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 37 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 38;

t) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 39 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 40;

u) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 41 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 42; or

v) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 44.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 45, 46, 47, 111, 112, and 113, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 2. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 1; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 2.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 48, 49, 50, 114, 115, and 116, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 3, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 4. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 4. 3; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 4.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 51, 52, 53, 117, 118, and 119, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 5, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 6. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 6. 5; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 6.

In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOS: 54, 55, 56, 120, 121 and 122, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 7, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 8. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 8. 7; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 8.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 57, 58, 59, 123, 124, and 125, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 9, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 10. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 10. 9; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 10.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 60, 61, 62, 126, 127, and 128, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 11, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 12. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 12. eleven; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 12.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 63, 64, 65, 129, 130, and 131, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 13, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 14. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 14. 13; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 14.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 66, 67, 68, 132, 133, and 134, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 15, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 16. 15; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 16.

In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, having the polypeptide sequences of SEQ ID NOS: 69, 70, 71, 135, 136 and 137,

- 13 043281 respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 17, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 18. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 18. 17; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 18.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 72, 73, 74, 138, 139, and 140, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 19, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 20. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 20. 19; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 20.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 75, 76, 77, 141, 142, and 143, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 21, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 22. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 22. 21; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 22.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 78, 79, 80, 144, 145, and 146, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 23, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 24. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 24. 23; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 24.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOS: 81, 82, 83, 147, 148, and 149, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 25, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 26. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 26. 25; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 26.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 84, 85, 86, 150, 151, and 152, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, contains a heavy chain variable region with a polypeptide sequence

- 14 043281 at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 27, and a light chain variable region having a polypeptide sequence of at least 85%, preferably 90 %, more preferably 95% identical to SEQ ID NO: 28. Preferably, the selected monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 27; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 28.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 87, 88, 89, 153, 154, and 155, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 29, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 30. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 30. 29; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 30.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 90, 91, 92, 156, 157, and 158, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 31, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 32. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 32. 31; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 32.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 93, 94, 95, 159, 160, and 161, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 33, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 34. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 34. 33; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 34.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 96, 97, 98, 162, 163, and 164, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 35, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 36. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 36. 35; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 36.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 99, 100, 101, 165, 166, and 167, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 37, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 38. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence

SEQ ID NO: 37; and a light chain variable region with a polypeptide sequence of SEQ ID NO:

38.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 102, 103, 104, 168, 169, and 170, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 39, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 40. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 40. 39; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 40.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 105, 106, 107, 171, 172, and 173, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 41, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 42. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 42. 41; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 42.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 108, 109, 110, 174, 175, and 176, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 43, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 44. Preferably, the isolated monoclonal antibody or antigen-binding fragment contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 44. 43; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 44.

In one embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 201, 202, 203, 204, 205, and 206, respectively. In another embodiment, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region with a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 199, and a light chain variable region having a polypeptide sequence at least 85%, preferably 90%, more preferably 95% identical to SEQ ID NO: 200. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof contains a heavy chain variable region with a polypeptide sequence of SEQ ID NO: 200. 199; and a light chain variable region with the polypeptide sequence of SEQ ID NO: 200.

According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigennegative fragment of the invention, where the antibody or antigennegative fragment is chimeric.

According to another specific aspect, the invention relates to an isolated monoclonal antibody or antigennegative fragment of the invention, where the antibody or antigennegative fragment is human or humanized.

In another specific aspect, the invention relates to an isolated humanized monoclonal antibody or antigen-binding fragment thereof according to the invention, wherein the isolated humanized antibody or antigen-binding fragment thereof comprises:

- 16 043281

a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

h) the heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and the light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

i) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

j) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 198;

k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 187 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 196;

n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 197; or

o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 199 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 200.

In another specific aspect, the invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is capable of blocking CD47 binding to thrombospondin-1 (TSP1) and/or signal regulatory protein alpha (SIRPa).

In another specific aspect, the invention relates to an isolated monoclonal antibody, or antigen-binding fragment thereof, wherein the antibody, or antigen-binding fragment thereof, is capable of inducing macrophage-mediated phagocytosis of cancer cells.

In another specific aspect, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is capable of binding to cancer cells with minimal to undetectable binding to erythrocytes. Binding of cancer cells by an isolated monoclonal antibody or antigen-binding fragment thereof according to the invention can be determined by methods known in the art.

In another general aspect, the invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment of the invention. Those skilled in the art will appreciate that the coding sequence of a protein can be changed (eg, by substitution, deletion, insertion, etc.) without changing the amino acid sequence of the protein. Accordingly, it will be appreciated by those skilled in the art that the nucleic acid sequences encoding the monoclonal antibodies or antigen-binding fragments thereof of the invention may be altered without altering the amino acid sequences of the proteins.

In another general aspect, the invention relates to a vector containing an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment of the invention. Any vector known to those skilled in the art in connection with the present invention may be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the implementation of the vector is a recombinant expression vector, such as a plasmid. The vector may include any element to provide the normal function of an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selectable marker, and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A number of expression vectors capable of delivering nucleic acids into a cell are known in the art and can be used according to the invention to produce an antibody or antigen-binding fragment thereof by a cell. Traditional methods of cloning or artificial gene synthesis can be used to create a recombinant expression vector in accordance with embodiments of the invention.

In another general aspect, the invention relates to a host cell containing an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment of the invention. Any host cell known to those skilled in the art in light of the present disclosure can be used to recombinantly express the antibodies or antigen-binding fragments thereof of the invention. In some embodiments, the host cells are E. coli TG1 or BL21 cells (for expressing, for example, scFv or Fab antibodies), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, for example, full length IgG antibodies) . In specific embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell's genome such that efficient expression of the recombinant nucleic acid is achieved.

In another general aspect, the invention relates to a method for producing a monoclonal antibody or an antigen-binding fragment thereof according to the invention, comprising culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof, under conditions for producing a monoclonal antibody or an antigen-binding fragment thereof, and isolating the antibody or antigen-binding fragment a fragment from a cell or culture (for example, from the supernatant). Expressed antibodies or antigen-binding fragments thereof can be collected from cells and purified in accordance with conventional methods known in the art and described in this application.

pharmaceutical compositions.

In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or an antigen-binding fragment thereof according to the invention and a pharmaceutically acceptable carrier. In the context of the present invention, the term pharmaceutical composition means a product containing an antibody of the invention together with a pharmaceutically acceptable carrier. The antibodies of the invention and compositions containing them are also useful in the manufacture of a medicament for the therapeutic applications mentioned herein.

As used herein, the term carrier refers to any excipient, diluent, excipient, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation shell, or other material well known in the art for use. in pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration used for the particular application. Used in the present description, the term pharmaceutically acceptable carrier refers to a non-toxic material that does not affect the effectiveness of the composition according to the invention or the biological activity of the composition according to the invention. In accordance with specific embodiments, in view of the present invention, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical composition of an antibody can be used in the invention.

The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, see for example The Science and Practice of Pharmacy (eg 21st edition (2005) and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, diluents, tonicity agents, preservatives, stabilizers, and chelating agents. In formulating the pharmaceutical compositions of the invention, one or more pharmaceutically acceptable carriers may be used.

In one of the embodiments of the invention, the pharmaceutical composition is a liquid composition. A preferred example of a liquid formulation is an aqueous formulation, i. composition containing water. The liquid formulation may contain a solution, suspension, emulsion, microemulsion, gel, and the like. The aqueous composition usually contains at least 50% wt./wt. water or at least 60, 70, 75, 80, 85, 90 or at least 95% wt./wt. water.

In one embodiment, the pharmaceutical composition may be formulated as an injectable solution, which can be administered, for example, using an injection device (eg, syringe or infusion pump). The injection may be, for example, subcutaneous, intramuscular, intraperitoneal, intravitreal or intravenous.

In another embodiment, the pharmaceutical composition is a solid formulation, such as a lyophilized or spray-dried composition, that can be used as is, or to which solvents and/or diluents are added by a clinician or patient prior to use. Solid dosage forms may include tablets, such as compressed and/or coated tablets, and capsules (eg, hard or soft gelatin capsules). The pharmaceutical composition may also be, for example, in the form of sachets, dragees, powders, granules, lozenges or lyophilized powders.

Dosage forms may be immediate release, in which case they may contain a water-soluble or dispersible carrier, or they may be delayed release, sustained release, or modified release, in which case they may contain water-insoluble polymers that regulate the rate of dissolution of the dosage form in the gastrointestinal tract or under the skin.

In other embodiments, the pharmaceutical composition may be delivered intranasally, buccally, or sublingually.

The pH of the aqueous composition may be from 3 to 10. In one embodiment, the pH of the composition is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the composition is from about 3.0 to about 7.0.

In another embodiment of the invention, the pharmaceutical composition contains a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid , tricine and tris(hydroxymethyl)aminomethane and mixtures thereof. The buffer may be present alone or as a mixture at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific buffers constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorcresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidourea, methyl 4hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal and mixtures thereof. The preservative may be present alone or as a mixture at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these specific preservatives constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a tonicity regulating agent. Non-limiting examples of this embodiment include salt (such as sodium chloride), amino acid (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditol (such as glycerol 1,2-propanediol, propylene glycol, 1 ,3-propanediol and 1,3-butanediol), polyethylene glycol (eg PEG400) and mixtures thereof. Another example of a tonicity agent includes sugar. Non-limiting examples of sugars can be mono-, di-, or polysaccharides or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha and beta. -HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethyl cellulose. Another example of a tonicity agent is sugar alcohol, where the term sugar alcohol is defined as a C(4-8) hydrocarbon having at least one OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. Pharmaceutical compositions each containing the tonicity-regulating agent referred to in this paragraph are alternative embodiments. The tonicity agent may be present alone or in admixture at a concentration of about 0.01 to about 50 mg/mL, such as about 0.1 to about 20 mg/mL. Pharmaceutical compositions containing each of these specific tonicity agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) salts, and mixtures thereof. The chelating agent may be present alone or in admixture at a concentration of from about 0.01 mg/mL to about 50 mg/mL, such as from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific chelating agents constitute alternative embodiments of the invention.

In another embodiment of the invention, the pharmaceutical composition contains a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment of the invention, the pharmaceutical composition contains a stabilizer, where the specified stabilizer is carboxy/hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, 2-methylthioethanol, polyethylene glycol (such as PEG 3350) , polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as monothioglycerol), or thioglycolic acid. The stabilizer may be present alone or in admixture at a concentration of from about 0.01 to about 50 mg/ml, such as from about 0.1 to about 20 mg/ml. Pharmaceutical compositions containing each of these particular stabilizers constitute alternative embodiments of the invention.

- 19 043281

In other embodiments, the pharmaceutical composition contains one or more surfactants, preferably a surfactant, at least one surfactant, or two different surfactants. The term surfactant refers to any molecules or ions that consist of a water-soluble (hydrophilic) part and a fat-soluble (lipophilic) part. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants and/or zwitterionic surfactants. The surfactant may be present alone or in admixture at a concentration of from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific surfactants constitute alternative embodiments of the invention.

In a further embodiment of the invention, the pharmaceutical composition contains one or more protease inhibitors such as, for example, EDTA and/or benzamidine hydrochloride (HCl). The protease inhibitor may be present alone or in admixture at a concentration of from about 0.1 mg/mL to about 20 mg/mL. Pharmaceutical compositions containing each of these specific protease inhibitors constitute alternative embodiments of the invention.

In another general aspect, the invention relates to a method for preparing a pharmaceutical composition containing a monoclonal antibody or antigen-binding fragment of the invention, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

Application methods.

In another general aspect, the invention relates to a method for blocking CD47 binding to thrombospondin-1 (TSP1) or a method for blocking CD47 binding to signal regulatory protein alpha (SIRPa), which method comprises administering to an individual a pharmaceutical composition of the invention.

The functional activity of antibodies and their antigen-binding fragments that bind to CD47 can be characterized by methods known in the art and described in this application. Methods for characterizing antibodies and antigen-binding fragments thereof that bind to CD47 include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, and OctetRed assays; ligand receptor binding assays to detect blocking of CD47 binding to TSP1 and/or SIRPa; phagocytosis assays in which CD47-expressing cells are labeled with a fluorescent dye and incubated with macrophages to detect the effect of blocking CD47 binding to SIRPa on phagocytosis of CD47-expressing cells by macrophages; hemagglutination assays to detect the effect of anti-CD47 antibodies on erythrocytes; and cell assays to detect the effect of blocking the TSP1-CD47 interaction on eNOS/NO/cGMP signaling further into endothelial cells. In specific embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind to CD47 include the methods described below.

In another general aspect, the invention relates to a method of treating cancer in a patient, comprising administering to an individual a pharmaceutical composition of the invention. The malignant tumor may be any hemoblastosis or solid cancer, for example, it may be selected from, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, cancer breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other types of hemoblastosis.

In another general aspect, the invention relates to a method of treating an inflammatory disease in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method of treating an infectious disease in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method of treating arteriosclerosis in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method of treating cardiovascular disease in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method for treating a metabolic disorder in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method for treating radiation injury in a patient, comprising administering to an individual a pharmaceutical composition of the invention.

In another general aspect, the invention relates to a method for treating an autoimmune disease in

- 20 043281 patient, including the introduction to the individual of the pharmaceutical composition according to the invention.

According to embodiments of the invention, the pharmaceutical composition contains a therapeutically effective amount of an anti-CD47 antibody or antigen-binding fragment thereof. Used in the present description, the term therapeutically effective amount refers to the amount of the active ingredient or component, which causes the desired biological or drug response in the individual. A therapeutically effective amount can be determined empirically and in a conventional manner depending on the stated purpose.

As used herein, a therapeutically effective amount with respect to an anti-CD47 antibody, or antigen-binding fragment thereof, means an amount of an anti-CD47 antibody, or antigen-binding fragment thereof, that modulates an immune response in a patient. Also, as used herein, a therapeutically effective amount with respect to an anti-CD47 antibody, or antigen-binding fragment thereof, means an amount of an anti-CD47 antibody, or antigen-binding fragment thereof, that results in the treatment of a disease, disorder, or condition; prevents or slows the progression of the disease, disorder or condition; or reduce or alleviate the symptoms associated with the disease, disorder or condition.

In specific embodiments, the disease, disorder, or condition being treated is a cancer, preferably a cancer selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urinary urothelial carcinoma. bladder, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblast leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other types of hemoblastosis. In other specific embodiments, the disease, disorder, or condition being treated is an inflammatory disease, an infectious disease, arteriosclerosis, a cardiovascular disease, a metabolic disorder, a radiation injury, an immune disease, and/or an autoimmune disease.

In specific embodiments, a therapeutically effective amount refers to an amount of a therapeutic agent that is sufficient to achieve one, two, three, four or more of the following effects: (i) reduce or alleviate the severity of the disease, disorder or condition being treated or associated with him a symptom; (ii) shortening the duration of the disease, disorder or condition being treated, or associated symptom; (iii) prevent progression of the disease, disorder or condition being treated, or associated symptom; (iv) inducing regression of the disease, disorder or condition being treated, or associated symptom; (v) preventing the development or onset of the disease, disorder or condition being treated, or a symptom associated therewith; (vi) preventing recurrence of the disease, disorder or condition being treated, or associated symptom; (vii) reducing the rate of hospitalization of an individual having a disease, disorder or condition being treated, or an associated symptom; (viii) reducing the duration of hospitalization of an individual suffering from a disease, disorder or condition being treated, or an associated symptom; (ix) increasing the survival of an individual suffering from a disease, disorder or condition being treated, or an associated symptom; (xi) inhibiting or alleviating the disease, disorder or condition being treated or associated symptom in an individual; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of the other therapy.

The therapeutically effective amount or dose may vary depending on various factors, such as the disease, disorder, or condition being treated, route of administration, target site, physiological state of the individual (including, for example, age, body weight, health), whether whether the individual is human or animal, other drugs administered, and the type of treatment: whether it is prophylactic or therapeutic. Therapeutic doses are selected so that optimal levels of safety and efficacy are achieved.

In specific embodiments, the compositions described herein are formulated into dosage forms suitable for the intended route of administration to the individual. For example, the compositions described herein may be formulated into a dosage form suitable for intravenous, subcutaneous, or intramuscular administration.

As used herein, the terms treat and treatment refer to the improvement or reversal of at least one measurable physical parameter associated with cancer, an immune disease, disorder or condition, an autoimmune disease, disorder or condition, or an inflammatory disease, disorder or condition, an infectious disease, disorder, or condition, arteriosclerotic disease, disorder or condition, cardiovascular disease, metabolic disorder or condition, metabolic disease, disorder or condition, radiation injury, disorder or condition

- 21 043281 em, which are not necessarily manifested, but can be manifested in an individual. The terms treat and treatment can also refer to regression, preventing the progression, or at least slowing down the progression of a disease, disorder or condition. In a particular embodiment, treating and treating refer to alleviating, preventing the development or onset of, or reducing the duration of, one or more symptoms associated with a disease, disorder, or condition, such as a tumor, or more preferably cancer. In a specific embodiment, treating and treating refer to preventing the recurrence of a disease, disorder, or condition. In a specific embodiment, treat and treat refer to increasing the survival of an individual suffering from a disease, disorder, or condition. In a specific embodiment, treating and treating refer to the elimination of a disease, disorder, or condition in an individual.

According to specific embodiments, a composition used in the treatment of cancer, an immune disease, disorder or condition, an autoimmune disease, disorder or condition, an inflammatory disease, disorder or condition, an infectious disease, disorder or condition, an arteriosclerotic disease, disorder or condition, a cardiovascular disease, metabolic disease, disorder or condition, radiation injury, disorder or condition may be used in combination with another type of treatment. For the treatment of cancer, the composition can be used in combination with another type of treatment, including, without limitation, chemotherapy, therapy with anti-CD20 mAb, anti-CTLA-4 antibodies, anti-LAG-3 mAb, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD33 mAb, anti-CD73 mAb, anti-CD47 mAb, anti-DLL-3 mAb, anti-apelin mAb, anti-TIP-1 mAb, anti-CLDN18.2 mAb, anti-FOLR1 mAb, anti-PD-L1 antibodies, anti-PD-1 antibodies, PD-1/PD-L1 therapy, or therapy with other immuno-oncological drugs, targeted therapy, therapy with anti-angiogenic agents, radiation therapy or therapy with other anti-cancer drugs. Ahtu-CD47 antibodies can be used to construct bispecific antibodies with mAb partners to PD-1, PD-L1, LAG3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, apelin, DLL3 , claudin 18.2, TIP-1, CD3, folate alpha receptor and/or other tumor surface antigens for the treatment of cancer/tumors that express both CD47 and tumor associated specific antigen.

As used herein, in combination, in the context of administering two or more therapeutic agents to an individual, refers to the use of more than one type of therapy. The use of the term in combination does not limit the order in which the therapeutic agents are administered to an individual. For example, the first therapeutic agent (for example, the composition described in this application) can be administered before (for example, 5, 15, 30, 45 minutes; 1, 2, 4, 6, 12, 16, 24, 48, 72, 96 hours ; 1, 2, 3, 4, 5, 6, 8, or 12 weeks), simultaneously or after (for example, after 5, 15, 30, 45 minutes; 1, 2, 4, 6, 12, 16, 24, 48 , 72, 96 hours; 1, 2, 3, 4, 5, 6, 8 or 12 weeks) administration of the second therapeutic agent to the individual.

In another general aspect, the invention relates to a method for determining the level of CD47 in an individual. The method includes (a) obtaining a sample from an individual; (b) bringing the sample into contact with the antibody or antigen-binding fragment of the invention; and (c) determining the level of CD47 in the individual.

As used herein, a sample refers to a biological sample obtained from an individual and may include, without limitation, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy (e.g., cancerous tissue, liver tissue, etc.) , lymphatic fluid, ascitic fluid, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucosa, sputum, sweat, urine, or any other secretions, excretion, or other body fluids. A blood sample refers to whole blood or any fraction thereof, including blood cells, serum and plasma. The blood sample may, for example, contain cancer cells.

In some embodiments, an individual's CD47 level can be determined using assays selected from, but not limited to, Western blot analysis, ELISA assay, FACS assay, and/or immunohistochemistry (IHC). Relative protein levels can be determined using Western blot analysis, FACS analysis and immunohistochemistry (IHC), and absolute protein levels can be determined using ELISA analysis. When determining relative CD47 levels, CD47 levels can be determined between at least two samples, e.g., between samples obtained from the same individual at different time points, between samples obtained from different tissues of the same individual, and/or between samples from different individuals. Alternatively, when determining absolute levels of CD47, for example using an ELISA assay, the absolute level of CD47 in a sample can be determined by creating a standard for the ELISA assay prior to testing the sample. One skilled in the art will understand which assay methods to use to determine the level of CD47 in a sample obtained from an individual using an antibody or antigen-binding fragment thereof of the invention.

The use of methods for determining the level of CD47 in a sample from an individual allows the diagnosis of abnormal (increased, decreased or insufficient) levels of CD47 in a disease to make appropriate decisions regarding therapy. Such a disease can be chosen, without

- 22 043281 restrictions, from a cancer, preferably a cancer selected from the group consisting of lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblast leukemia (ALL), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML) , multiple myeloma (MM), acute myeloid leukemia (AML) and other types of hemoblastosis, inflammatory disease, infectious disease, arteriosclerosis, cardiovascular disease, metabolic disorder, radiation injury, immune disease and/or autoimmune disease.

Embodiments

The invention also provides for the following non-limiting embodiments.

Embodiment 1 is an isolated monoclonal antibody, or an antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 having the polypeptide sequences:

(1) SEQ ID NOs: 177, 46, 47, 178, 112 and 179, respectively;

(2) SEQ ID NOs: 51, 52, 53, 117, 118 and 119, respectively;

(3) SEQ ID NOs: 54, 55, 56, 120, 121 and 122, respectively;

(4) SEQ ID NOs: 57, 58, 59, 123, 124 and 125, respectively;

(5) SEQ ID NOs: 60, 61, 62, 126, 127 and 128, respectively;

(6) SEQ ID NOs: 180, 181, 182, 129, 130 and 131, respectively;

(7) SEQ ID NOs: 72, 73, 74, 138, 139 and 140, respectively;

(8) SEQ ID NOs: 78, 79, 80, 144, 145 and 146, respectively;

(9) SEQ ID NOs: 81, 82, 83, 147, 148 and 149, respectively;

(10) SEQ ID NOs: 84, 85, 86, 150, 151 and 152, respectively;

(11) SEQ ID NOs: 87, 88, 89, 153, 154 and 155, respectively;

(12) SEQ ID NOs: 90, 91, 92, 156, 157 and 158, respectively;

(13) SEQ ID NOs: 93, 94, 95, 159, 160 and 161, respectively;

(14) SEQ ID NOs: 96, 97, 98, 162, 163 and 164, respectively;

(15) SEQ ID NOs: 99, 100, 101, 165, 166 and 167, respectively;

(16) SEQ ID NOs: 102, 103, 104, 168, 169 and 170, respectively;

(17) SEQ ID NOs: 105, 106, 107, 171, 172 and 173, respectively;

(18) SEQ ID NOs: 108, 109, 110, 174, 175 and 176, respectively; or (19) SEQ ID NOs: 201, 202, 203, 204, 205 and 206, respectively;

wherein the antibody, or antigen-binding fragment thereof, specifically binds to CD47, preferably human CD47.

Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment of Embodiment 1 comprising a heavy chain variable region with a polypeptide sequence that is at least 95% identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 , 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43, or a light chain variable region with a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or 44.

Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment of Embodiment 1 or 2, comprising:

(a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 2;

(b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 3 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 4;

(c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 5 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 6;

(d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 7 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 8;

(e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 9 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 10;

(f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 11 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 12;

(g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 13 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 14;

(h) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 16;

(i) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 17 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 18;

- 23 043281 (j) the heavy chain variable region with the polypeptide sequence of SEQ ID NO: 19 and the light chain variable region with the polypeptide sequence of SEQ ID NO: 20;

(k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 21 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 22;

(l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 23 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 24;

(m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 25 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 26;

(n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 27 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 28;

(o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 30;

(p) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 31 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 32;

(q) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 33 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 34;

(r) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 35 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 36;

(s) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 37 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 38;

(t) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 39 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 40;

(u) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 41 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 42; or (v) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 44.

Embodiment 4 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-3, wherein the antibody or antigen-binding fragment thereof inhibits the interaction of CD47 with thrombospondin-1 (TSP1) and/or CD47 with SIRPa.

Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-4, wherein the antibody or antigen-binding fragment thereof is chimeric.

Embodiment 6 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-5, wherein the antibody or antigen-binding fragment thereof is human or humanized.

Embodiment 7 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 6, wherein the antibody or antigen-binding fragment thereof comprises:

a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

c) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

d) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

e) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 192;

f) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 184 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

g) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 190;

h) the heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and the light chain variable region with the polypeptide sequence of SEQ ID NO: 191;

i) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 193;

j) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 185 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 198;

k) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 187 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

l) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and

- 24 043281 light chain variable region with the polypeptide sequence of SEQ ID NO: 194;

m) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 196;

n) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 188 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 197; or

o) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 199 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 200.

Embodiment 8 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-7, wherein the antibody or antigen-binding fragment thereof is capable of blocking CD47 binding to thrombospondin-1 (TSP1) and/or signal regulatory protein SIRPa alpha.

Embodiment 9 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-7, wherein the antibody or antigen-binding fragment thereof is capable of inducing macrophage-mediated phagocytosis of cancer cells.

Embodiment 10 is an isolated monoclonal antibody or antigen-binding fragment according to any one of Embodiments 1-7, wherein the antibody or antigen-binding fragment thereof is capable of binding to cancer cells with minimal to undetectable binding to erythrocytes.

Embodiment 11 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any one of Embodiments 1-10.

Embodiment 12 is a vector containing the isolated nucleic acid of Embodiment 11.

Embodiment 13 is a host cell containing the vector of Embodiment 12.

Embodiment 14 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment of any one of Embodiments 1-10 and a pharmaceutically acceptable carrier.

Embodiment 15 is a method of blocking CD47 binding to thrombospondin-1 (TSP1) in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 16 is a method of blocking CD47 binding to signal regulatory protein alpha (SIRPa) in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 17 is a method for treating cancer in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 18 is a method of treating an inflammatory disease in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 19 is a method for treating an infectious disease in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 20 is a method for treating arteriosclerosis in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 21 is a method for treating cardiovascular disease in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 22 is a method for treating a metabolic disorder in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 23 is a method for treating radiation injury in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 24 is a method of treating an autoimmune disease in a patient, comprising administering to the patient the pharmaceutical composition of Embodiment 14.

Embodiment 25 is a method for determining a CD47 level in an individual, comprising (a) obtaining a sample from the individual; (b) bringing the sample into contact with an antibody or antigen-binding fragment according to any one of embodiments 1-10; and (c) determining the level of CD47 in the individual.

Embodiment 26 is the method of Embodiment 25 wherein the sample is a tissue sample.

Embodiment 27 is the method of Embodiment 26 wherein the tissue sample is a cancer tissue sample.

- 25 043281

Embodiment 28 is the method of Embodiment 25 wherein the sample is a blood sample.

Embodiment 29 is a method for producing a monoclonal antibody or an antigen-binding fragment according to any one of Embodiments 1-10, comprising culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment under conditions to produce a monoclonal antibody or an antigen-binding fragment, and isolating the antibody or an antigen-binding fragment from a cell or culture.

Embodiment 30 is a method for preparing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment of any one of Embodiments 1-10, comprising combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

Examples

Example 1 Identification of monoclonal anti-CD47 antibodies.

Monoclonal anti-CD47 antibodies (mAb) were obtained from mice immunized with recombinant human CD47-HIS and java poppy. Briefly, after immunization, serum antibody titer was assessed by ELISA using huCD47-HIS and cyCD47-HIS coated plates. B cells were harvested and fused with a myeloma cell line to form hybridomas. Hybridomas were plated in 20 x 384 well plates and the supernatants from each well were screened by ELISA for binding to human and cynomolgus CD47. 400 hybridomas were propagated and further analyzed for binding to RAJI cells by FACS assay, blocking of CD47/SIRPa interaction, binding kinetics to recombinant huCD47 by Octet system, and tested for hemagglutination activity using human blood. The best positive hybridomas were then cloned by seeding the parental hybridomas 1 cell per well in 384 well plates and screening the clonal supernatants by ELISA for binding to huCD47. Heavy chain and light chain variable regions derived from clonal hybridomas were amplified with 5'RACE and sequenced. The supernatants of these clones from the scale-up culture were used to purify the Protein A antibodies for further characterization.

The sequences of the variable regions of the heavy and light chains of monoclonal CD47 antibodies are presented in table. 1 and 2, respectively, a CDR region of monoclonal anti-CD47 antibodies are presented in table. 3 and 4. CDR regions of monoclonal anti-CD47 antibodies were determined using the IMGT method.

- 26 043281

Sequences in;

Table 1 heavy chain regions of monoclonal aHmu-CD47 antibodies (mAb)

mAb clones vh 9O23A QIQLQQSGPELVRPGASVKISCKASGYTFTDYYINWVKQRPGQGLEWIG WIYPGSGNTKYNEKFKGKATLTVDSSSSTAYMQLSSLTSEDSAVYFCAR RGPWYFDVWGAGTTVTVSS (SEQ ID NO:1) 14P6A QIQLQQSGPELVRPGASVKISCKASGYTFTAYYINWVKQRPGQGLEWIG WIYPGSGNTKYNEKFKGKATLTVDTSSSTAYIQLSSLTSEDSAVYFCAR RGPWYFDVWGAGTTVTVSS (SEQ ID NO:3) 4M8A QVQLQQPGAELVKPGASVKLSCKTSGYTFTSYWIHWVNQRPGQGLEWI GNIDPSDSETHYNPKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCA RWGGWLPLDYWGQGTTLTVSS (SEQ ID NO:5) 16M17A QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYY CARMAFITTVVDYWGQGTTLTVSS (SEQ ID NO:7) 13С4А QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYY CARWGYYGRSPLDHWGQGTTLTVSS (SEQ ID NO:9) 14O18A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTLDKSSSTAYMQLSSLTSEDSAVYY CARWYYGGSGAMDYWGQGTSVTVSS (SEQ ID NO: 11) 5D24A QVQLQQPGAELVKPGASVKLSCKASGYTFTSSWMHWVKQRPGQGLEW IGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYC ARWGYYGKSAIDYWGQGTSVTVSS (SEQ ID NO:13) 11G2A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE

- 27 043281

WIGNIDPSDSEAHYNQKFRDKATLTVDKSSSTAYMQLSSLTSEDSAVYY CARWGYYGKSAMDYWGQGTSVTVSS (SEQ ID NO: 15) 13V18A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYY CSRWGYYGKSAMDYWGQGTSVTVSS (SEQ ID NO: 17) 1J7A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFRDKATLTVDKSSNTAYMQLSSLTSEDSAVYY CARWGLRGAMDYWGQGTSVTVS (SEQ ID NO: 19) 14D18A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYY CARWGYYGRSPLDHWGQGTTLTVSS (SEQ ID NO:21) 305A EVKLVESGGGLVQSGRSLRLSCATSGFTFSDFYMEWVRQAPGKGLEWI AASRNKANDYTTEYSASVKGRFIVSRDTSQSILYLQMNALRAEDTAIYY CARDTAYWGQGTLVTVSA (SEQ ID NO:23) 17С6А QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLE WIGNIDPSDSETHYNQKFKDKATLTVDKSSSTAHMQLSSLTSEDSAVYY CAGTDLAYWGQGTLVTVSA (SEQ ID NO:25) 14N13A QVQLQQPGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEW IGNIDPSDSETHYNQKFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYC AKGFSDYWGQGTTLTVSS (SEQ ID NO:27) 10I23A EVQLQQSGAELVRPGASVKLSCTASGFNIKDSLMHWVKQRPEQGLEWI GWIDPEDGETKCAPKFQDKATITADTSSNTAYLQLSSLTSEDTAIYYCAV ISTVVAPDYWGQGTTLTVSS (SEQ ID NO:29) 12V18A EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWI GRINPYNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLTSEDSAIYYCA RGGVVATDYWGQGTTLTVSS (SEQ ID NO:31) 17O12A EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMHWVKQSHGKSLEWI GRINPYNGDTFNNQKFKGKATLAVDKSSSTAHMELRSLTSEDSTVYYC ARGGYAMDYWGQGTSVTVSS (SEQ ID NO:33) 15G23A EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKQKPGQGLEWI GYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCA KGGTGTGDYWGQGTTLTVSS (SEQ ID NO:35) 17N8A EVKLEESGGGMVQPGGSMKVSCVASGFTFSNYWMNWVRQSPEKGLE WVAQIRLKSDNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTG IYYCTGGGKGGFAYWGQGTLVTVSA (SEQ ID NO:37) 18M19A EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEW IGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYC AKGGYYAMDYWGQGTSVTVSS (SEQ ID NO:39) 11F6A EVQLQQSGAELVRPGASVKLSCTASGFNIKDSLMHWVKQRPEQGLEWI GWIDPEDGETKCAPKFQDKATITADTSSNTAYLQLSSLTSEDTAIYYCAR ITTVVATDYWGQGTTLTVSS (SEQ ID NO:41) 19L14A QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWI GNSNPGSSSTNYNEKFKSKAILTVDKSSSTAYMQLSSLTSDDSAVYYCA REGLRRFAYWGQGTLVTVSA (SEQ ID NO:43)

VH: heavy chain variable region.

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table 2

Light chain variable region sequences of monoclonal aHmu-CD47 mAb

mAb clones VL 9O23A NIVMTQSPKSMSMSVGERVTLSCKASDNVGTYVSWYQQKPEQSPKLLI YGASNRYTGVPDRFTGSGSARDFTLTITSVQAEDLADYHCGQSYSYPLT FGAGTKLELK (SEQ ID NO:2) 14P6A NIVMTQSPKSMSMSVGERVTLSCKASENVGTYVSWYQQKPEQSPNLLIY GASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQTYSYPLTF GAGTKLELK (SEQ ID NO:4) 4M8A DVQITQSPSYLAASPGETITINCRASKNISKYLAWFQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISRLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO:6) 16M17A DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO:8) 13С4А DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG SSLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO: 10) 14O18A DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO: 12) 5D24A DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG

- 29 043281

STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO: 14) 11G2A DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO: 16) 13V18A AVQITQFPSYLAASPGQTITINCRASKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO: 18) 1J7A DVQITQSPTYLTASPGETITINCRANKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO:20) 14D18A DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSG STLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGG TKLEIK (SEQ ID NO:22) 305A DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQ LLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEY PFTFGSGTKLEIK (SEQ ID NO:24) 17С6А DIQMNQSPSSLSASLGDTITITCHASQNINVWLSWYQQKPGNIPKLLIYK ASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPWTFGG GTKLEIK (SEQ ID NO:26) 14N13A DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQ SPKVLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQY YSYPLTFGAGTKLELK (SEQ ID NO:28) 10I23A DVVMTQTPLSLPVSLGVQASISCRSSQSLVHSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PWTFGGGTKLEIK (SEQ ID NO:30) 12V18A DVVMTQTPVSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PFTFGSGTKLEIK (SEQ ID NO:32) 17O12A DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLYWYLQKPGQSP KLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCFQSTHV PHTFGGGTKLEIK (SEQ ID NO:34) 15G23A DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PPLTFGAGTKLELK (SEQ ID NO:36) 17N8A DVVMTQTPLSLPVSLGDQASISCRSTQSLVHSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PLTFGAGTKLELK (SEQ ID NO:38) 18M19A DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PWTFGGGTKLEIK (SEQ ID NO:40) 11F6A DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSP KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHV PLTFGAGTKLELK (SEQ ID NO:42) 19L14A ENVLTQSPAIMSASPGEKVTMTCRASSSVSSSYLHWYQQKSGASPKLWI YSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPFTF GSGTKLEIK (SEQ ID NO:44)

VL: light chain variable region.

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Table 3

aHTu-CD47 mAb heavy chain region 1-3 CDR

mAb clones HC CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) 9O23A GYTFTDYY (45) IYPGSGNT (46) ARRGPWYFDV(47) 14P6A GYTFTAYY (48) IYPGSGNT (49) ARRGPWYFDV(50) 4M8A GYTFTSYW(51) IDPSDSET (52) ARWGGWLPLDY(53) 16M17A GYTFTNYW(54) IDPSDSET (55) ARMAFITTVVDY(56) 13C4A GYTFTSYW (57) IDPSDSET (58) ARWGYYGRSPLDH(59) 14O18A GYTFTSYW (60) IDPSDSET (61) ARWYYGGSGAMDY (62) 5D24A GYTFTSW(63) IDPSDSET (64) ARWGYYGKSAIDY (65) 11G2A GYTFTSYW (66) IDPSDSEA (67) ARWGYYGKSAMDY (68) 13B18A GYTFTSYW(69) IDPSDSET (70) SRWGYYGKSAMDY(71) 1J7A GYTFTSYW(72) IDPSDSET (73) ARWGLRGAMDY (74) 14D18A GYTFTSYW (75) IDPSDSET (76) ARWGYYGRSPLDH (77) 3O5A GFTFSDFY(78) SRNKANDYTT (79) ARDTAY (80) 17C6A GYTFTSYW(81) IDPSDSET (82) AGTDLAY (83) 14N13A GYIFTSYW (84) IDPSDSET (85) AKGFSDY(86) 10I23A GFNIKDSL(87) IDPEDGET (88) AVISTVVAPDY(89) 12B18A GYSFTGYF (90) INPYNGDT (91) ARGGVVATDY (92) 17O12A GYSFTGYF(93) INPYNGDT (94) ARGGYAMDY (95) 15G23A GYTFTNYV (96) INPYNDGT (97) AKGGTGTGDY (98) 17N8A GFTFSNYW(99) IRLKSDNYAT (100) TGGGKGGFAY(lOl) 18M19A GYTFTSYV (102) INPYNDGT (103) AKGGYYAMDY (104) 11F6A GFNIKDSL (105) IDPEDGET (106) ARITTVVATDY (107) 19L14A GYTFTSYW (108) SNPGSSST (109) AREGLRRFAY(llO)

HC: heavy chain; CDR: complementarity determining region CDR of HC anti-CD47 mAb was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).

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Table 4

Light chain CDR region 1-3 aHmu-CD47 mAb

LC clones mAb CDR1 (SEQ ID NO:) CDR2 (SEQID CDR3 (SEQ ID NO:) NO:) 9O23A DNVGTY (111) GAS (112) GQSYSYPLT (113) 14P6A ENVGTY(114) GAS (115) GQTYSYPLT (116) 4M8A KNISKY(117) SGS(118) QQHNEYPWT (119) 16M17A KSISKY (120) SGS(121) QQHNEYPWT (122) 13C4A KSISKY (123) SGS(124) QQHNEYPWT (125) 14O18A KSISKY (126) SGS(127) QQHNEYPWT (128) 5D24A KSISKY (129) SGS(130) QQHNEYPWT (131) 11G2A KSISKY (132) SGS(133) QQHNEYPWT (134) 13B18A KSISKY (135) SGS(136) QQHNEYPWT (137) 1J7A KSISKY (138) SGS(139) QQHNEYPWT (140) 14D18A KSISKY (141) SGS(142) QQHNEYPWT (143) 3O5A KSLLHSNGNTY (144) RMS (145) MQHLEYPFT (146) 17C6A QNINVW(147) KAS (148) QQGQSYPWT (149) 14N13A QSLLYSSNQKNY WAS (151) QQYYSYPLT (152) (150) 10I23A QSLVHSNGNTY (153) KVS (154) SQSTHVPWT (155) 12V18A QSLVHSNGNTY (156) KVS (157) SQSTHVPFT (158) 17O12A QSLVHSNGNTY (159) RVS (160) FQSTHVPHT (161) 15G23A QSLVHSNGNTY (162) KVS (163) SQSTHVPPLT (164) 17N8A QSLVHSNGNTY (165) KVS (166) SQSTHVPLT (167) 18M19A QSLVHSNGNTY (168) KVS (169) SQSTHVPWT (170) 11F6A QSLVHSNGNTY (171) KVS (172) SQSTHVPLT (173) 19L14A SSVSSSY (174) STS (175) QQYSGYPFT (176)

LC: light chain; CDR: complementarity determining region.

The CDR LC of the anti-CD47 mAb was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Res. 1999; 27: 209-212).

Example 2 Detection of CD47 mAb binding to RAJI cells by FACS analysis.

Ahtu-CD47 mAbs were analyzed by flow cytometry for their ability to bind to CD47 on the cell surface. RAJI cells (ATCC #CCL-86) (20,000 cells) cultured in Hanks' Balanced Salt Solution (HBSS) were incubated with either a solution of purified mAbs (1 μg/ml) in HBSS or HBSS alone. Using a secondary IgG Ab conjugated to AlexaFluor488, the presence of mouse anti-CD47 mAbs on RAJI cells was measured using FACS (IntelliCyt iQue® Screener; Albuquerque, NM). The results of the FACS analysis of anti-CD47 mAb binding are shown in FIG. 1.

Example 3 Assay for the ability of an anti-CD47 mAb to block the CD47/SIRPa interaction.

The activity of hybridoma supernatants or purified anti-CD47 mAbs to block the SIRPa/CD47 interaction was measured by ELISA assay. Recombinant human CD47(ECD)-HIS (1 μg/ml) was immobilized on a 384-well ELISA plate. Binding of recombinant huSIRP-huFc-biotin protein (final concentration 0.5 μg/ml) was evaluated in the presence of increasing amounts of mouse anti-CD47-mAbs in triplicate. Bound SIRPa was determined using an HRP-conjugated secondary antibody complexed with streptavidin (Thermo Fisher Scientific; Waltham, MA). After addition of CD47, blocking solution, SIRPa protein, secondary antibody and detection reagents, washing steps were performed using PBS supplemented with 0.1% Tween20. The results of the ELISA assays are shown in FIG. 2A-2O. For the 18M19A chimeric antibody (IgG4 and cap backbone) (FIG. 2P), blocking activity was measured in a 96-well ELISA plate. Recombinant human CD47(ECD)-HIS (1 μg/ml) was immobilized on the plate. Binding of recombinant huSIRPa-muFc (final concentration 0.5 μg/ml) was measured in the presence of increasing amounts of human anti-CD47 mAb in duplicate. Bound SIRPa was measured with an HRP-conjugated secondary anti-muFc antibody (Jackson ImmunoResearch; West Grove,

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PA). The plates were washed as described above. Detection was performed using a substrate

TMB for detection (Thermo Fisher Scientific; Waltham, MA). The result of the ELISA assay is shown in FIG. 2p.

Example 4 Evaluation of the ability of an anti-CD47 mAb to induce hemagglutination.

To evaluate the hemagglutination capacity of anti-CD47 mAbs, purified mAbs were added at 2-fold serial dilution to a 96-well transparent round bottom plate and incubated with a 2.5% suspension of human erythrocytes (huRBC) in PBS (final RBC concentration 0.25%) at room temperature in for 1 hour. The absence of hemagglutination was indicated by the presence of clear spots in the center of the round bottom plates. Evidence of hemagglutination was the presence of a uniform color throughout the well. The results of the haemagglutination assay are shown in FIG. 3A-3B.

Example 5 In Vivo Evaluation of the Efficacy of Chimeric 13B18A in a RAJI Cell Xenograft Model.

A chimeric variant of the 13B18A antibody (13B18A-huIgG1) was constructed by fusing the variable regions (VH and VL) of 13B18A with human kappa heavy chain and light chain constant regions, respectively. The resulting antibody was stably expressed in stable CHO pools and purified using a protein A affinity column. To evaluate the potency of 13B18A-huIgG1, RAJI cells were inoculated subcutaneously in the right flank of each NOD/SCID mouse (female, u = 10/group). Mice were treated with the indicated doses of test antibodies when the average tumor size reached 100 mm ³ . Doses were administered intravenously 3 times a week from days 6 to 27, and tumor volumes were measured on the same day in two dimensions using a caliper. For pharmacokinetic (PK) analysis, serum was collected 48 hours after the final dose and serum levels of 13B18A-huIgG1 were measured by detection of human Fc bound on recombinant human CD47(ECD)-HIS-coated ELISA plates. A standard curve was built using serum labeled with 13B18A-huIgG1 as a standard with a known concentration. After washing with PBS supplemented with 0.1% Tween-20, bound antibody was detected with an HRP-conjugated secondary anti-huFc antibody (Jackson ImmunoResearch, West Grove, PA) followed by the addition of TMB detection substrate (Thermo Fisher Scientific; Waltham, MA) . Tumor growth curves are shown in Fig. 4A. Body weight data is shown in Fig. 4b. Pharmacokinetic parameters (PK) are shown in FIG. 4C.

Example 6 Humanization of anti-CD47 mAb.

The mouse anti-CD47 mAbs 13B18A, 14P6A and 17C6A were humanized to reduce immunogenicity when used in human patients. The sequences of the heavy and light chain variable regions (VH and VL) were compared with human antibody sequences present in the Protein Data Bank (PDB) and homology models were built. The CDRs of both heavy and light chains of mouse mAbs were grafted onto human scaffolds that had the highest probability of retaining the proper structure likely required for antigen binding. If necessary, backmutate from human residues to mouse residues or other mutations. The sequence of humanized VH and VL regions are given in table. 5 and 6, respectively. The humanized VH and VL regions were fused to human IgG4 heavy chain and kappa light chain constant regions, respectively. Constructs corresponding to mAb sequences were used for transient transfection into 293E cells and purified mAbs were analyzed by ELISA for their ability to block the SIRPa/CD47 interaction. The results are presented as optical density, where higher optical density indicates a higher level of SIRPa/CD47 interaction. IC ₅₀ values for humanized mAbs are shown in Table 1. 7. IC ₅₀ curves for humanized H3L9 and H5L5 mAbs are shown in FIG. 5A-5B. The results of the haemagglutination assay are shown in FIG. 6. The CDR regions of the humanized mAb H8L10 are shown in Table 1. 8.

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Table 5

Heavy chain variable region sequences of humanized aHmu-CD47 mAb

Constructions I vh SEQID NO: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHW VRQAPGQG H1 LEWMGNIDPSDSETHYNQKFKDRVTTLTVDTSTSTVYME 183 LSSLRSEDT AVYYCSRWGYYGKSAMDYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHW VRQAPGQGL H2 EWMGNIDPSDSETHYNQKFKDRVTLTVDTSTSTAYMEL 184 SSLRSEDTA VYYCSRWGYYGKSAMDYWGQGTLVTVSS NZ QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHW VRQAPGQGL EWIGNIDPSDSETHYNQKFKDRATLTVDTSTSTAYMELS SLRSEDTA VYYCSRWGYYGKSAMDYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHW VRQRPGQGL 185 H4 EWIGNIDPSDSETHYNQKFKDRATLTVDTSTSTAYMELS SLRSEDTA VYYCSRWGYYGKSAMDYWGQGTLVTVSS QIQLVQSGAEVKKPGASVKVSCKASGYTFTAYYINWVR QAPGQRLE 186 H5 WIGWIYPGSGNTKYNEKFKGRVTLTVDTSASTAYIELSS LRSEDTAV YYCARRGPWYFDVWGQGTTVTVSS QIQLVQSGAEVKKPGASVKVSCKASGYTFTAYYINWVR QAPGQRLE 187 H6 WIGWIYPGSGNTKYNEKFKGRVTLTVDTSASTAYIELSS LRSEDTAV YFCARRGPWYFDVWGQGTTVTVSS QIQLVQSGAEVKKPGASVKISCKASGYTFTAYYINWVR QAPGQGLE 188 H7 WIGWIYPGSGNTKYNEKFKGRATLTVDTSASTAYIELSS 189 LRSEDTAV YFCARRGPWYFDVWGQGTTVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHW VRQAPGQG H8 LEWIGNIDPSDSETHYAQKFQGRVTTLTVDKSTSTVYME 199 LSSLRSEDT AVYYCAGTDLAYWGQGTLVTVSS

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Table 6

Humanized anti-CD47 mAb light chain variable region sequences

Construction VL SEQ ID NO: L1 AVQLTQSPSFLSASVGDRVTITCRASKSISKYLAWYQQKP 190 L2 L3 L4 L5 L6 L7 GKANKLLIYSG STLQSGVPSRFSGSGSGTEFTLTISSLQPEDFAMYYCQQH NEYPWTFGGGT KVEIK AVQLTQSPSFLSASVGQRITINCRASKSISKYLAWYQQKP GKANKLLIYSGS TLQSGVPSRFSGSGSGTEFTLTISSLQPEDFAMYYCQQHN EYPWTFGGGTK VEIK AVQLTQSPSFLSASVGQRITINCRASKSISKYLAWYQEKP GKANKLLIYSGS TLQSGIPSRFSGSGSGTDFTLTISSLQPEDFAMYYCQQHN EYPWTFGGGTK VEIK AVQITQSPSFLSASVGQTITINCRASKSISKYLAWYQEKPG KANKLLIYSGST LQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNE YPWTFGGGTKVE IK EIVMTQSPATLSLSPGERATLSCRASENVGTYVSWYQQK PGQAPNLLIYGA SNRYTGIPARFSGSGSGTDFTLTISSLQPEDFAVYHCGQT YSYPLTFGQGTK LEIK EIVMTQSPATLSLSPGERATLSCRASENVGTYVSWYQQK PGQSPNLLIYGA SNRYTGIPDRFSGSGSGTDFTLTISSLQPEDFAVYHCGQT YSYPLTFGQGTK LEIK EIVMTQSPATLSLSPGERATLSCKASENVGTYVSWYQQK PGQSPNLLIYGA SNRYTGIPDRFSGSGSGTDFTLTISSLQPEDFAVYHCGQT YSYPLTFGQGTK LEIK 191 192 193 194 195 196 L8 NIVMTQSPATLSLSPGERATLSCKASENVGTYVSWYQQK 197

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PGQSPNLLIYGA SNRYTGVPDRFSGSGSATDFTLTISSLQPEDFADYHCGQT YSYPLTFGQGTK LEIK L9 DVQLTQSPSFLSASVGDRVTITCRASKSISKYLAWYQQKP GKANKLLIYSG STLQSGVPSRFSGSGSGTEFTLTISSLQPEDFAMYYCQQH NEYPWTFGGGT KVEIK 198 L10 EIVMTQSPGTLSLSPGERATLSCHASQNINVWLSWYQQK PGQAPRLLIYKA SNLHTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQG QSYPFTFGQGTK VEIK 200

Table 7

IC ₅₀ values for anti-CP47 TL in the SIRPa/CD47 interaction assay

mAb ID IC50 (nM) H1L2 6.80 H1L3 7.43 H1L4 8.09 H2L1 4.73 H2L3 9.56 H2L4 6.52 H3L1 5.62 H3L2 7.88 H3L4 5.35 H3L9 9.60 H5L5 15.06 H6L5 9.67 H6L7 39.16 H6L8 13.20 H8L10 1.05

H1L2 refers to mAb with an H1 heavy chain variable region and an L2 light chain variable region; these rules correspond to all other humanized SHAL given in the table.

Table 8

Humanized rnAb H8L10 heavy and light chain CDR regions 1-3

CDR1 (SEQ ID NO:) CDR2 (SEQ ID NO:) CDR3 (SEQ ID NO:) NA GYTFTSYW(201) IDPSDSET (202) AGTDLAY (203) LC QNINVW(204) KAS (205) QQGQSYPFT(206)

Example 7 RBC and RAJI Binding Assays.

Using flow cytometry, the ability of anti-SE47 mAb H3L9, H5L5 and H8L10 to bind to CD47 on the cell surface was analyzed. A serial dilution (1:3) of the purified mAbs was performed in FACS buffer (Hanks Balanced Salt Solution (HBSS) containing 0.1% BSA and 0.05% sodium azide). The upper concentration of the purified mAb was 190 μg/ml. Erythrocytes or RAJI cells (ATCC #CCL-86) (14,000 cells) were pelleted in U-shaped dishes by centrifugation at 600 rpm (62xg) for 5 min. Cells were resuspended in 20 µl of purified antibodies in FACS buffer and incubated for 30 min at room temperature. After incubation, cells were washed three times with FACS buffer. Using a secondary PE/Cy7-conjugated Ab to human IgG (Biolegend, Cat # 409316), the amount of anti-CE47 mAb on erythrocytes and RAJI cells was measured by FACS analysis (Attune NxT flow cytometer; Carlsbad, CA). The results of the FACS analysis for anti-CD47 mAb binding are shown in FIG. 7A-7B.

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Example 8 Cellular SIRPa Binding Assay.

RAJI cells were cultured in RPMI+10% FBS. Human SIRPa(ECD)-mFc protein (huSIRPamuFc) (human SIRPa ECD fused to mouse Fc) at a final concentration of 30 nM was incubated with purified humanized anti-CD47 mAbs at 30, 90 and 300 nM. The mixture was then added to 14,000 RAJI cells in a round bottom 96 well plate, mixed and incubated in a nutator for 30 minutes at room temperature. Cells were then centrifuged at 600 rpm for 5 min and washed three times with FACS buffer (HBSS supplemented with 0.1% BSA and 0.05% sodium azide). Cells were then incubated with FITC-conjugated polyclonal donkey anti-mouse Fc antibodies (Jackson ImmunoResearch, Cat: 715-095-150) in a nutator for 15 min at room temperature, washed three times with FACS buffer, and then resuspended in FACS buffer. The cells were then passed through the Attune NxT instrument and the data was analyzed using the Attune NxT software. The results of humanized anti-CD47 mAbs H3L9, H5L5 and H8L10 blocking huSIRPa-muFc binding to RAJI cells are shown in FIG. 8.

Example 9 Analysis of macrophage mediated phagocytosis.

Human monocytes were induced for 6 days in AIM-V medium (Thermo Fisher, Cat: 12055091) containing 50 ng/ml GM-CSF (Shenandoah, Cat: 100-08-20ug). Macrophages were then polarized with 100 ng/ml INF-gamma (Shenandoah, Cat: 100-77-100 μg) for an additional 2 days. M1 macrophages were defined as a population of CD14+, CD80+, CD163 ^- and CD206+ cells. After separation from tissue culture plates, macrophages were washed once with RPMI-1640 containing 10% FBS and then twice with ice-cold HBSS. The number of cells was adjusted to 2x106 cells/ml in AIM-V medium. To 50 µl of CFSE-labeled RAJI cells (100,000 cells (Thermo Fisher, Cat: 34570) in each well of a 96-well plate, 25 µl of test mAb and 25 µl of macrophage cell suspension (50,000 cells) were added and incubated for 2 hours at 37°C. The final mAb concentration was 10 μg/ml. After co-culture, cell mixtures were stained with PE-Cy7-conjugated human CD14 mAb (Biolegend, Cat: 367111). After staining, the cells were analyzed by flow cytometry. The percentage of both CFSE and PE-Cy7 positive macrophages in a population of PE-Cy7 positive macrophages is presented as phagocytosis. The results of humanized anti-CD47 mAb H3L9, H5L5, and H8L10 induction of macrophage-mediated phagocytosis of RAJI cells are shown in FIG. 9.

Those skilled in the art will appreciate that changes may be made to the embodiments described above without deviating from the general concept of the invention. Therefore, it is intended that the present invention is not limited to the specific disclosed embodiments, and covers modifications falling within the spirit and scope of the present invention as defined by the present description.

Claims (18)

1. An isolated monoclonal antibody or its antigen-binding fragment containing complementarity determining region 1 of the heavy chain (HCDR1), HCDR2, HCDR3, complementarity determining region 1 of the light chain (LCDR1), LCDR2 and LCDR3 with polypeptide sequences: (1) SEQ ID NOs: 201, 202, 203, 204, 205 and 206, respectively; or (2) SEQ ID NOs: 81, 82, 83, 147, 148, and 149, respectively; where the antibody or antigennegative fragment specifically binds to CD47. 2. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein CD47 is human CD47. 3. An isolated monoclonal antibody or antigen-binding fragment according to claim 1 or 2, containing a heavy chain variable region with a polypeptide sequence that is at least 95% identical to SEQ ID NO: 199 or 25, or a light chain variable region with a polypeptide sequence that at least 95% identical to SEQ ID NO: 200 or 26. 4. An isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, containing: a) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 199 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 200; or b) a heavy chain variable region with the polypeptide sequence of SEQ ID NO: 25 and a light chain variable region with the polypeptide sequence of SEQ ID NO: 26. 5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is chimeric or human or humanized. 6. The isolated monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is capable of blocking CD47 binding to signal-regulatory protein alpha (SIRPa), inducing macrophage-mediated phagocytosis of malignant cells and/or binding to malignant cells, when this binding to erythrocytes pro- - 37 043281 emanates at a level from minimal to undetectable. 7. A bispecific antibody comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1-6. 8. An isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-6. 9. An isolated nucleic acid encoding a bispecific antibody according to claim 7. 10. An expression vector containing the isolated nucleic acid according to claim 8 or 9. 11. A host cell containing the vector of claim 10. 12. Pharmaceutical composition containing selected monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-6 and a pharmaceutically acceptable carrier or bispecific antibody according to claim 7 and a pharmaceutically acceptable carrier. 13. A method for blocking CD47 binding to signal regulatory protein a (SIRPa) in a patient, comprising administering to the patient a pharmaceutical composition according to claim 12. 14. A method for determining the level of CD47 in an individual, including: (a) obtaining a sample from an individual; (b) bringing the sample into contact with the antibody or antigen-binding fragment according to any one of claims 1-6; and (c) determining the level of CD47 in the individual. 15. The method of claim 14, wherein the sample is a tissue sample or a blood sample, optionally wherein the tissue sample is a cancerous tissue sample. 16. A method for producing a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, comprising culturing a cell containing a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof under conditions for producing a monoclonal antibody or an antigen-binding fragment thereof and isolating the antibody or its an antigen-binding fragment from a cell or culture. 17. A method for producing a pharmaceutical composition containing a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 6, including combining the monoclonal antibody or antigen-binding fragment with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. 18. A method of treating a disease and/or condition selected from the group consisting of a malignant tumor, an inflammatory disease, an infectious disease, arteriosclerosis, a cardiovascular disease, a metabolic disorder, a radiation injury, and an autoimmune disease in a patient, comprising administering to the patient a pharmaceutical composition according to claim 12 .