HK40013319B - Anti-cd47 antibody and use thereof - Google Patents

Description

anti-CD 47 antibodies and uses thereof

The present invention relates to novel antibodies and antibody fragments that specifically bind to integrin-associated proteins (IAPs), (also referred to as CD 47) and compositions containing the same. The invention also relates to nucleic acids encoding the antibodies or antibody fragments thereof, and host cells comprising the same, as well as related uses. Furthermore, the invention also relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Background

Cancer immunotherapy is a serious trend in the field of bioscience in recent years, and immune checkpoint inhibitor therapy using T-cell-based CTLA4 antibody, PD-1 antibody, PD-L1 antibody, and other cell therapy such as CAR-T, TCR-T are recently high-grade immunotherapies. These are nothing else but around how to restore T cell function, in other words, mainly around how to boost the capacity of the adaptive immune system. However, the approach of targeting immune checkpoints (checkpoint) to activate T cell function and thus increase the ability of the acquired immune system to overcome cancer is still complicated. However, the role of the innate immune system in tumor immunotherapy has not been played for a long time. In fact, macrophages account for about 50% of the tumor tissue in the whole tumor infiltration area, and more importantly, the number of macrophages is inversely related to the prognosis of the tumor, which further indicates that macrophages have a very important role in the tumor.

Two signals are required for the phagocytic effect of macrophages to act simultaneously: one is the activation of the "eat-me" signal targeting the cell surface, and the other is the inactivation of the "do-me" signal of the same target surface. The absence of either signal is insufficient to trigger the phagocytic effect to occur. There is increasing evidence that CD47 is a class of "eat-me" signals that inhibit phagocytosis by macrophages by interacting with Signal regulatory protein alpha (SIRP α) on the surface of macrophages. Tumor cells can also evade macrophage phagocytosis through the expression of CD47 (see, e.g., EP2242512 and related references cited therein).

CD47, also known as integrin-associated protein (IAP), is a member of the immunoglobulin superfamily. CD47 is widely expressed on the cell surface, and can interact with SIRP alpha, thrombospondin-1, TSP1 and integrin to mediate a series of reactions such as apoptosis, proliferation and immunity. TSP1 is involved in cell proliferation, growth and differentiation, and the binding of CD47 to TSP1 plays an important role in regulating cell migration, cell proliferation and apoptosis, as well as promoting angiogenesis and inflammatory responses. In addition, CD47 is an important self-recognition marker on the cell surface. CD47 binds to macrophage surface sirpa, phosphorylates its Immunoreceptor Tyrosine Inhibitory Motif (ITIM), and subsequently recruits SHP-1 protein, resulting in a cascade of reactions that inhibit phagocytosis of macrophages (see, e.g., US9382320 and related references cited therein).

Different studies have shown that almost all tumor cells and tissues highly express CD47. CD47 highly expressed on the surface of the tumor cells is combined with SIRP alpha on the surface of macrophages to release a 'do not eat me' signal, so that the macrophages in a tumor tissue infiltration area are harmonious with the tumor cells, and the macrophage in the tumor tissue infiltration area can inhibit T-cell effect by promoting the proliferation of blood vessels in the tumor, thereby promoting the proliferation and growth of the tumor cells.

The role of CD47 in promoting cell proliferation depends to a large extent on the cell type, as activation and loss of CD47 can lead to enhanced proliferation. Activation of CD47 with TSP-1 increased proliferation of human U87 and U373 astrocytoma cells, but not normal astrocytes. In addition, the CD47 blocking antibody inhibited the proliferation of unstimulated astrocytoma cells, but not normal astrocytes. Although the exact mechanism is not clear, CD47 may promote cancer cell proliferation through the PI3K/Akt pathway, but not normal cell proliferation (Sick E, boukhari A, deramaudt, rond Wep, bucher B, andr P, gies JP, takeda K., activation of CD47 receptors promotion of human astrocytoma but not normal assays via an Akt-dependent pathway, glia.2011 Feb,59 (2): 308-19.

CD47 ligation results in cell death of many normal and tumor cell lines by apoptosis or autophagy. Activation of CD47 induces rapid apoptosis of T cells. Jurkat cells and Peripheral Blood Mononuclear Cells (PBMCs) incubated with monoclonal antibody Ad22 caused apoptosis within 3 hours. However, no apoptosis was observed after incubation with other anti-CD 47 antibodies. The apoptosis-inducing function of CD47 appears to be dependent on the activation of specific epitopes on the extracellular domain (Pettersen RD, hestdal K, olafsen MK, lie SO, lindberg FP (June 1999), CD47 signals T cell death, J.Immunol.162 (12): 7031-40.PMID 10358145).

A number of anti-CD 47 antibodies have been reported so far, for example US2015/0183874 A1 reports a chimeric monoclonal antibody of human IgG1 type derived from B6H12 and a humanized B6H12 antibody produced by CDR grafting, which has lower immunogenicity compared to known antibodies. US 9045541 reports an anti-CD 47 antibody that does not significantly cause hemagglutination, and which is significantly effective in tumor models, e.g. increasing the ability of macrophages to phagocytose tumor cells, compared to known antibodies.

Most antibodies known in the prior art that resist the binding of CD47 to SIRPa promote phagocytosis of macrophages while causing agglutination of red blood cells, thereby compromising the therapeutic effect of the corresponding antibodies.

Therefore, in the treatment of various tumors and/or cancers, there is still an urgent need to develop an anti-CD 47 antibody having good target specificity, excellent therapeutic effects (e.g., improving phagocytosis of macrophages, inhibiting tumor growth, and even causing complete disappearance of tumors), and less side effects. The present invention meets this need.

Summary of The Invention

The invention provides anti-CD 47 antibodies, compositions, kits, methods and uses related to anti-CD 47 antibodies.

The inventors of the present invention surprisingly found that the antibody developed by the present invention has significant anti-tumor activity, can significantly inhibit the growth of tumor, and even can completely eliminate tumor.

In some embodiments, the invention provides anti-CD 47 antibodies or antibody fragments (preferably antigen-binding fragments) that bind CD47 or fragments thereof (preferably human CD47 protein).

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise or consist of one to three heavy chain complementarity determining regions HCDRs selected from the group consisting of seq id nos: (i) HCDR1 comprising a sequence selected from SEQ ID NO: 1.2, 3, 4, 5,6, 7, 8, 98 and 99, (ii) an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9. 10, 11, 12, 13, 14, 15, 16, 100 and 101, (iii) an HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 17. 18, 19, 20, 21, 22, 102 and 103, and (iv) HCDRs of (i), (ii) and (iii) that contain at least one and no more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions, the anti-CD 47 antibody comprising the modified CDR still has the ability to bind CD47.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise or consist of one to three light chain complementarity determining regions, LCDRs, selected from the group consisting of seq id nos: (i) LCDR1 comprising SEQ ID NO:23 or 24, (ii) an LCDR2 comprising the amino acid sequence of SEQ ID NO:25 or 26, (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27. 28, 29 or 30, and (iv) an LCDR of (i), (ii) or (iii) that contains at least one and no more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions, the anti-CD 47 antibody comprising the modified CDR still has the ability to bind CD47.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise a) one to three heavy chain complementarity determining regions HCDRs selected from the group consisting of: (i) a HCDR1 comprising a sequence selected from SEQ ID NO: 1.2, 3, 4, 5,6, 7, 8, 98 and 99, (ii) an HCDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9. 10, 11, 12, 13, 14, 15, 16, 100 and 101, (iii) a HCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 17. 18, 19, 20, 21, 22, 102 and 103, (iv) HCDRs of (i), (ii) and (iii) that contain at least one and no more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions; and B) one to three light chain complementarity determining regions (LCDRs) selected from the group consisting of: (i) LCDR1 comprising SEQ ID NO:23 or 24, (ii) LCDR2 comprising the amino acid sequence of SEQ ID NO:25 or 26, (iii) an LCDR3 comprising the amino acid sequence of SEQ ID NO: 27. 28, 29 or 30, and (iv) an LCDR of (i), (ii) or (iii) that contains at least one and no more than 5 amino acid substitutions (e.g., conservative substitutions), deletions or insertions, the anti-CD 47 antibody comprising the modified CDR still has the ability to bind CD47.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 1.2, 3, 4, 5,6, 7, 8, 98 and 99, or consists of said amino acid sequence; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 9. 10, 11, 12, 13, 14, 15, 16, 100 and 101, or consists of said amino acid sequence; the HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 17. 18, 19, 20, 21, 22, 102 and 103 or consists of said amino acid sequence.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 23 or 24 or consists of said amino acid sequence; LCDR2 comprises an amino acid sequence selected from SEO ID NO:25 and 26 or consists of said amino acid sequence; LCDR3 comprises a sequence selected from SEQ ID NO: 27. 28, 29 and 30 or consists of said amino acid sequence.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the invention comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 1.2, 3, 4, 5,6, 7, 8, 98 and 99, or consists of said amino acid sequence; the HCDR2 comprises an amino acid sequence selected from SEQ ID NO: 9. 10, 11, 12, 13, 14, 15, 16, 100 and 101, or consists of said amino acid sequence; the HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 17. 18, 19, 20, 21, 22, 102 and 103 or consists of said amino acid sequence; LCDR1 comprises an amino acid sequence selected from SEO ID NO:23 and 24 or consists of said amino acid sequence; LCDR2 comprises an amino acid sequence selected from SEQ ID NO:25 and 26 or consists of said amino acid sequence; LCDR3 comprises a sequence selected from SEQ ID NO: 27. 28, 29 and 30 or consists of said amino acid sequence.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:98 or 99, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:100 or 101, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:102 or 103 or consists thereof; wherein LCDR1 comprises an amino acid sequence selected from SEQ ID NO:23 and 24, and LCDR2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:25 and 26, and LCDR3 comprises or consists of an amino acid sequence selected from SEO ID NO: 27. 28, 29 and 30 or consists of said sequence.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:1 or consists thereof; HCDR2 comprises SEQ ID NO:9 or consists thereof; HCDR3 comprises SEQ ID NO:17 or consists thereof; LCDR1 comprises SEQ ID NO:23 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEQ ID NO:27 or consists of the amino acid sequence shown in seq id no.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises SEQ ID NO:2 or consists of the amino acid sequence shown in the specification; HCDR2 comprises SEQ ID NO:10 or consists thereof; HCDR3 comprises SEQ ID NO:18 or consists thereof; LCDR1 comprises SEQ ID NO:23 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEQ ID NO:27 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:3 or consists thereof; HCDR2 comprises SEQ ID NO:11 or consists thereof; HCDR3 comprises SEQ ID NO:17 or consists thereof; LCDR1 comprises SEQ ID NO:23 or consists thereof; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEQ ID NO:27 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises SEQ ID NO:1 or consists thereof; HCDR2 comprises SEQ ID NO:9 or consists of the amino acid sequence shown in the formula (I); HCDR3 comprises SEQ ID NO:19 or consists of the amino acid sequence shown in seq id no; LCDR1 comprises SEQ ID NO:23 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEO ID NO:28 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:4 or consists thereof; HCDR2 comprises SEQ ID NO:9 or consists of the amino acid sequence shown in the formula (I); HCDR3 comprises SEQ ID NO:19 or consists thereof; LCDR1 comprises SEQ ID NO:23 or consists thereof; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEQ ID NO:28 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the HCDR1 comprises SEQ ID NO:5 or consists thereof; HCDR2 comprises SEQ ID NO:12 or consists thereof; HCDR3 comprises SEQ ID NO:19 or consists of the amino acid sequence shown in seq id no; LCDR1 comprises SEQ ID NO:23 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:25 or consists thereof; and LCDR3 comprises SEQ ID NO:28 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:6 or consists of the amino acid sequence shown in the specification; HCDR2 comprises SEQ ID NO:13 or consists thereof; HCDR3 comprises SEO ID NO:20 or consists thereof; LCDR1 comprises SEQ ID NO:24 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:26 or consists of the amino acid sequence shown in seq id no; and LCDR3 comprises SEQ ID NO:29 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:7 or consists thereof; HCDR2 comprises SEQ ID NO:14 or consists of an amino acid sequence set forth in seq id no; HCDR3 comprises SEQ ID NO:20 or consists thereof; LCDR1 comprises SEQ ID NO:24 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:26 or consists of the amino acid sequence shown in seq id no; and LCDR3 comprises SEQ ID NO:29 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:8 or consists of the amino acid sequence shown in seq id no; HCDR2 comprises SEQ ID NO:15 or consists thereof; HCDR3 comprises SEQ ID NO:21 or consists of the amino acid sequence shown in 21; LCDR1 comprises SEQ ID NO:24 or consists thereof; LCDR2 comprises SEQ ID NO:26 or consists of the amino acid sequence shown in seq id no; and LCDR3 comprises SEQ ID NO:30 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein HCDR1 comprises SEQ ID NO:7 or consists thereof; HCDR2 comprises SEQ ID NO:16 or consists thereof; HCDR3 comprises SEQ ID NO:22 or consists thereof; LCDR1 comprises SEQ ID NO:24 or consists of the amino acid sequence shown in seq id no; LCDR2 comprises SEQ ID NO:26 or consists of the amino acid sequence shown in seq id no; and LCDR3 comprises SEQ ID NO:30 or consists thereof.

In some embodiments, an anti-CD 47 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region HCVR comprising an amino acid sequence that hybridizes to a sequence selected from the group consisting of SEO ID NO: 44. 45, 46, 47, 48, 49, 50, 51, 52 and 53, or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical to said sequence. In some embodiments, the heavy chain variable region HCVR of the anti-CD 47 antibody comprises a HCVR amino acid sequence that is identical to a sequence selected from SEQ ID NOs: 44. 45, 46, 47, 48, 49, 50, 51, 52, and 53, has one or more substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence, but an anti-CD 47 antibody comprising the HCVR has the ability to bind CD47.

In some embodiments, an anti-CD 47 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region LCVR comprising a sequence identical to SEQ ID NO: 54. 55, 57 and 58 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity. In some embodiments, the light chain variable region LCVR of the anti-CD 47 antibody comprises a sequence identical to SEQ ID NO: 54. 55, 57, and 58 with one or more substitutions (e.g., conservative substitutions), insertions, or deletions, but an anti-CD 47 antibody comprising the LCVR has the ability to bind CD47.

In some embodiments, an anti-CD 47 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises an amino acid sequence that is identical to a sequence selected from SEQ ID NOs: 44. 45, 46, 47, 48, 49, 50, 51, 52 and 53, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity; the light chain variable region LCVR comprises an amino acid sequence substantially identical to SEQ ID NO: 54. 55, 57 and 58 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:44 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:54 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:45 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:54 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:46 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:54 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises SEQ ID NO:47 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:55 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:48 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:55 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises SEQ ID NO:49 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:55 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises SEQ ID NO:50 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:57 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:51 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:57 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:52 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:58 or consists thereof.

In a preferred embodiment, the invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:53 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:58 or consisting of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 74. 76, 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97, or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical to said sequence. In some embodiments, the heavy chain of the anti-CD 47 antibody comprises a heavy chain identical to a heavy chain selected from SEQ ID NOs: 74. 76, 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97, but which comprises the heavy chain has the ability to bind CD47.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO: 75. 79, 83 or 86, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity. In some embodiments, the light chain of the anti-CD 47 antibody comprises a heavy chain variable region comprising a heavy chain variable region sequence identical to a light chain variable region selected from SEQ ID NOs: 75. 79, 83 and 86 with one or more substitutions (e.g. conservative substitutions), insertions or deletions compared to the amino acid sequence set forth above, but an anti-CD 47 antibody comprising said light chain has the ability to bind CD47.

In some embodiments, the anti-CD 47 antibodies, or antigen-binding fragments thereof, of the invention comprise a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 74. 76, 77, 78, 80, 81, 82, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 and 97, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence; the light chain comprises a sequence identical to SEQ ID NO: 75. 79, 83, 86, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:74 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:76 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:77 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:78 or consists thereof; the light chain comprises SEQ ID NO:79, or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:80 or consists thereof; the light chain comprises SEQ ID NO:79 or consist thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:81 or consists thereof; the light chain comprises SEQ ID NO:79 or consist thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:82 or consists thereof; the light chain comprises SEQ ID NO:83 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:84 or consists thereof; the light chain comprises SEQ ID NO:83 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:85 or consists thereof; the light chain comprises SEQ ID NO:86 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:87 or consists thereof; the light chain comprises SEQ ID NO:86 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:88 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:89 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:90 or consists thereof; the light chain comprises SEQ ID NO:75 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:91 or consists of the amino acid sequence shown in seq id no; the light chain comprises SEQ ID NO:79, or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:92 or consists thereof; the light chain comprises SEQ ID NO:79 or consist thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:93 or consists thereof; the light chain comprises SEQ ID NO:79, or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:94 or consists thereof; the light chain comprises SEQ ID NO:83 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:95 or consists thereof; the light chain comprises SEQ ID NO:83 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:96 or consists thereof; the light chain comprises SEQ ID NO:86 or consists thereof.

In a preferred embodiment, the present invention provides an anti-CD 47 antibody or antigen-binding fragment thereof, wherein the heavy chain comprises SEQ ID NO:97 or consists thereof; the light chain comprises SEQ ID NO:86 or consists thereof.

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of an anti-CD 47 antibody, antibodies that compete with any of the antibodies described above for binding to CD47, and antibodies that bind to the same epitope of CD47 as any of the antibodies described above.

In some embodiments, the anti-CD 47 antibody is a monoclonal antibody. In some embodiments, the anti-CD 47 antibody is humanized. In some embodiments, the anti-CD 47 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-CD 47 antibody is a human consensus framework sequence. In one embodiment, the anti-CD 47 antibodies of the invention also encompass antibody fragments thereof, preferably antibody fragments selected from the group consisting of: fab, fab '-SH, fv, scFv or (Fab') ₂ And (3) fragment.

In some embodiments, the anti-CD 47 antibodies of the invention are blocking antibodies that block the binding of CD47 to sirpa.

In one aspect, the invention provides nucleic acids encoding any of the above anti-CD 47 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one embodiment, the invention provides a method of making an anti-CD 47 antibody or fragment thereof (preferably an antigen-binding fragment), wherein said method comprises culturing said host cell under conditions suitable for expression of a nucleic acid encoding said antibody or fragment thereof (preferably an antigen-binding fragment), and optionally isolating said antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-CD 47 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In one embodiment, the invention provides anti-CD 47 antibodies or fragments thereof made by the methods of the invention.

In some embodiments, the present invention provides a composition comprising any of the anti-CD 47 antibodies or fragments thereof (preferably antigen-binding fragments thereof) described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In one aspect, the invention relates to a method of inhibiting CD47 binding to sirpa in a subject, the method comprising administering to the subject an effective amount of any anti-CD 47 antibody or fragment thereof herein. The invention also relates to the use of any of the anti-CD 47 antibodies or fragments thereof disclosed herein to prepare a composition or medicament for inhibiting CD47 binding to sirpa in a subject.

In one aspect, the invention relates to a method of promoting phagocytosis of macrophages in a subject, the method comprising administering to the subject an effective amount of an anti-CD 47 antibody or fragment thereof any herein. The invention also relates to the use of any of the anti-CD 47 antibodies or fragments thereof disclosed herein for the preparation of a composition or medicament for promoting phagocytosis of macrophages in a subject. In one embodiment, the anti-CD 47 antibodies of the invention can increase phagocytosis of macrophages by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to a control.

In another aspect, the invention relates to a method of treating a CD 47-associated disorder in a subject, comprising administering to the subject an effective amount of any of the anti-CD 47 antibodies or fragments thereof herein. The invention also relates to the use of any of the anti-CD 47 antibodies or fragments thereof herein for the manufacture of a medicament for treating a CD 47-associated disorder in a subject.

In some embodiments, the CD 47-associated disorder is a variety of hematological disorders and solid tumors, including but not limited to Acute Myelogenous Leukemia (AML), chronic myelogenous leukemia, acute Lymphocytic Leukemia (ALL), non-hodgkin lymphoma (NHL), multiple Myeloma (MM), lymphoma, breast cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, renal cancer, pancreatic cancer, bladder cancer, glioma, melanoma, and other solid tumors.

In one aspect, the invention relates to a method of CD 47-targeted tumor immunotherapy comprising administering to a subject an effective amount of any of the anti-CD 47 antibodies or fragments thereof described herein. The invention also relates to the use of any of the anti-CD 47 antibodies or fragments thereof disclosed herein for the manufacture of a medicament for the treatment of a tumor.

In one aspect, the invention relates to methods of treating any disease or disorder that may be ameliorated, alleviated, inhibited or prevented by the elimination, inhibition or reduction of CD47 activity.

In another aspect, the methods of the invention also relate to methods of treating a tumor by combination therapy, comprising administering to a subject an effective amount of any of the anti-CD 47 antibodies or fragments thereof described herein and one or more additional agents. In some embodiments, the methods disclosed herein further comprise co-administering to the subject an effective amount of a second medicament, wherein the anti-CD 47 antibody or fragment thereof disclosed herein is the first medicament. In one embodiment, the second drug is a chemotherapeutic agent, a radiotherapeutic agent, or a biomacromolecule drug for treating a related disease. In one embodiment, the biomacromolecule drug is, for example, a variety of monoclonal antibody drugs that attack tumor cells through T cell recognition, such as rituximab, cetuximab, and trastuzumab. The expression "second drug" as used herein does not mean that it refers to the only drug other than the first drug. Thus, the second drug need not be one drug, but may constitute or comprise more than one such drug.

In some embodiments, the subject or individual is a mammal, preferably a human.

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof provided herein are effective in promoting phagocytosis by macrophages.

In a preferred embodiment, the anti-CD 47 antibodies or antigen-binding fragments thereof provided herein are surprisingly effective in inhibiting tumor growth as compared to a control antibody.

In a more preferred embodiment, the anti-CD 47 antibodies or antigen-binding fragments thereof provided by the present invention are capable of completely regressing tumors, which was completely unexpected and has never been reported in the prior art.

In one aspect, the invention relates to a method of detecting CD47 protein in a sample, comprising (a) contacting the sample with any of the anti-CD 47 antibodies or fragments thereof described herein; and (b) detecting the formation of a complex between the anti-CD 47 antibody or fragment thereof and the CD47 protein. In certain embodiments, CD47 is human CD47. In one embodiment, the detection method may be an in vitro or in vivo method. In one embodiment, the anti-CD 47 antibody is used to select a subject suitable for treatment with the anti-CD 47 antibody. In one embodiment, the anti-CD 47 antibody is detectably labeled.

In another aspect, the invention relates to a method of determining the effectiveness of a cancer therapy comprising the step of measuring the number of CD47 expressing cancer cells in a sample from a subject before and after treatment, a decrease in CD47 expressing cancer cells after treatment indicating that the therapy is effective.

The invention also encompasses any combination of any of the embodiments described herein. Any embodiment described herein or any combination thereof is applicable to any and all anti-CD 47 antibodies or fragments, methods and uses thereof of the invention described herein.

Description of the drawings:

FIG. 1 cell level affinity detection of anti-CD 47 antibodies in IgG1 format produced in yeast by flow cytometry.

FIG. 2 flow cytometry determined blockade of SIRPa binding to CD47 expressed on CHO cells by antibodies of the invention in the form of IgG1 produced in yeast cells.

FIG. 3 flow cytometry generated in CHO cells blocks binding of SIRPa to CD47 expressed on CHO cells by antibodies of the invention in the form of IgG 4.

FIG. 4 detection of the ability of the IgG1 form of the antibodies of the invention produced in yeast cells to promote phagocytosis of tumor cells by macrophages includes antibodies ADI-29336, ADI-29340, ADI-29341, and ADI-29349 after affinity maturation of the IgG1 form of the antibodies produced in yeast cells.

FIG. 5 detection of the ability of macrophages to phagocytose tumor cells by antibodies of the invention to IgG1 produced in yeast cells.

FIG. 6 is a graph showing the ability of the antibody of the present invention in the form of IgG4 produced in CHO cells to promote phagocytosis of tumor cells by macrophages.

FIG. 7 detection of the ability of the antibody of the invention in the form of IgG4 produced in CHO cells to promote phagocytosis of tumor cells by macrophages. Including ADI-29336, ADI-29340, ADI-29341, and ADI-29349 and ADI-29371.

FIG. 8: anti-tumor Activity of the antibody ADI-26630 of the invention in the form of IgG4 produced in CHO cells in a mouse model (NOD/SCID Raji tumor model). Wherein fig. 8B is a partial enlarged view of fig. 8A.

FIG. 9: anti-tumor Activity of the antibody ADI-26624 of the invention in the form of IgG4 produced in CHO cells in a mouse model (NOD/SCID Raji tumor model).

FIG. 10: anti-tumor Activity of the antibodies of the invention ADI-26630, ADI29340 and ADI29341 in the form of IgG4 produced in CHO cells in the mouse model (NOD/SCID Raji tumor model) was studied at a dose of 0.5mg/kg.

FIG. 11: anti-tumor Activity of the antibodies of the invention ADI-26630, ADI29340 and ADI29341 in the form of IgG4 produced in CHO cells in a mouse model (NOD/SCID Raji tumor model) was studied at a dose of 5mg/kg.

FIG. 12: the anti-CD 47 antibody of the invention promotes the result of the activity detection of hemagglutination.

Detailed Description

1.1 definition of

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include the plural and vice versa, as appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about," when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

The term "conservative substitution" refers to the substitution of one amino acid with another amino acid within the same class, e.g., the substitution of one acidic amino acid with another acidic amino acid, the substitution of one basic amino acid with another basic amino acid, or the substitution of one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table:

the term "antibody" is used herein in the broadest sense and encompasses a variety of antibody constructs, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. A complete antibody will typically comprise at least two full length heavy chains and two full length light chains, but in some cases may comprise fewer chains, for example an antibody naturally occurring in a camel may comprise only heavy chains.

The term "antigen-binding moiety" as used herein refers to a moiety that specifically binds to a target antigen. The term includes antibodies that are capable of specifically binding to a target antigen as well as other natural (e.g., receptor, ligand) or synthetic molecules (e.g., darpins). In a preferred embodiment, the antigen binding portion of the antibody of the invention is an antibody fragment.

The terms "full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single amino acid composition, and not to the method of production thereof. The monoclonal antibody or antigen-binding fragment thereof can be, for example, generated by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.

As used herein, the terms "bind" and "specifically bind" refer to the binding of an antibody or antigen-binding portion to an epitope in an in vitro assay, preferably in a biophotonic interferometry with purified wild-type antigen (ForteBio). In certain embodiments, an antibody or antigen-binding portion is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

Antibodies are classified in "classes" depending on the amino acid sequence of their heavy chain constant region: igA, igD, igE, igG, and IgM, and several of these classes can be further divided into subclasses, e.g., igG1, igG2, igG3, and IgG4, igA1, and IgA2. The heavy chain constant regions corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. The light chain constant regions (CL) that can be found in all five antibody classes are called κ and λ. Within full-length light and heavy chains, the variable and constant regions are typically connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, e.g., fundamental Immunology, ch.7 (Paul, w. Editions, second edition, raven Press, n.y. (1989)) which is incorporated herein by reference in its entirety for all purposes. The variable region of each light/heavy chain pair typically forms an antigen binding site.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three complementarity determining regions. (see, e.g., kindt et al Kuby Immunology,6 ^th ed., w.h.freeman and co.91 page (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., portolano et al, j.immunol.150:880-887 (1993); clarkson et al, nature 352:624-628 (1991).

The variable regions typically exhibit the same general structure of relatively conserved Framework Regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from both chains of each pair are typically positioned (align) by framework regions, which allow binding to a specific epitope. The two light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.

A "complementarity determining region" or "CDR" or "hypervariable region" (used interchangeably herein with hypervariable region "HVR") is the region of amino acids in an antibody variable region which is primarily responsible for binding to an antigenic epitope. The CDRs of the heavy and light chains are generally referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3.

Various protocols for determining the CDR sequences of a given VH or VL amino acid sequence are known in the art: kabat Complementarity Determining Regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, md. (1991)), while Chothia refers to the position of structural loops (Chothia et al, (1987) J.Mol.biol.196:901-917 Chothia et al (1989) Nature 342-877-883), abM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software, and "Contact" (Contact) HVR is based on analysis of complex crystal structures available. The residues of each of these HVRs/CDR are as follows, according to different CDR determination schemes.

In one embodiment, the CDRs of an antibody of the invention are CDR sequences located at the following Kabat residue positions according to the Kabat numbering system:

positions 24-34 (LCDR 1), 50-56 (LCDR 2), and 89-97 (LCDR 3) in VL, and positions 27-35 (HCDR 1), 50-65 (HCDR 2), and 93-102 (HCDR 3) in VH.

CDRs may also be determined based on the same Kabat numbering position as a reference CDR sequence (e.g., any one of the exemplary CDRs of the invention).

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

The term "competition" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between the antigen binding proteins, as determined by the following assay: in such assays, the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., CD47 or a fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assays (see, e.g., stahli et al, 1983, methods in Enzymology 9. Typically the assay involves the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be detected is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring. Additional details regarding methods for determining competitive binding are provided in the examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., decrease) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of a reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source, using a human antibody library or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the choice of human immunoglobulin VL or VH sequences is selected from a subset of variable domain sequences. In general, a subtype of this sequence is a subtype as in Kabat et al, sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, bethesda MD (1991), volumes 1-3. In one embodiment, for VL, this subtype is subtype kappa I as in Kabat et al (see above). In one embodiment, for the VH, this subtype is subtype III as in Kabat et al (supra).

"humanized" antibodies are chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are more fully described in e.g. EP 404,097; WO 1993/01161; hudson et al, nat. Med.9:129-134 (2003); and Hollinger et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The terms "effective amount," "therapeutically effective amount" refers to an amount or dose of an antibody or antigen-binding fragment of the invention that, upon administration to a patient in a single or multiple doses, produces the desired effect in the treated subject, which includes improvement of the subject's condition (e.g., improvement of one or more symptoms) and/or delay in progression of the symptoms, and the like. An "effective amount" or "therapeutically effective amount" may also refer to an amount sufficient to reduce CD47 signaling (see, e.g., yamauchi et al, 2013 blood, jan 4.; soto-Pantoja et al, 2013 Expert Opin target, 17.

In one embodiment, an effective amount of a CD47 antibody of the invention can promote/increase phagocytosis of macrophages by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to a control.

An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors: species such as mammals; its size, age and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.

As described above, in some cases, the interaction between an antibody and its target antigen can interfere with the function of the target. The amount required for administration further depends on the binding affinity of the antibody for its specific antigen, and also on the rate of clearance of the administered antibody in the subject receiving the administration. By way of non-limiting example, a typical range of therapeutically effective doses of an antibody or antibody fragment of the invention is from about o.1mg/kg body weight to about 100mg/kg body weight. In some embodiments, an antibody of the invention is administered to a subject at a dose of 0.1mg/kg, 0.5mg/kg, 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, 15mg/kg, 20mg/kg, 25mg/kg, 30mg/kg, 50mg/kg, 75mg/kg, 100mg/kg or more. Common dosage frequency ranges are, for example, twice daily to once weekly, once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every half year.

The term "block" as used herein means reduced CD47 signaling in the presence of an antibody of the invention. Blocking of CD 47-mediated signaling refers to a decrease in the level of CD47 signaling in the presence of a CD47 antibody of the invention by an amount greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% below the control level of CD47 (i.e., the level of CD47 signaling in the absence of the antibody). The level of CD47 signaling can be measured using a variety of standard techniques, such as, by way of non-limiting example, measuring downstream gene activation and/or luciferase reporter assays in response to CD47 activation. One skilled in the art will appreciate that CD47 signaling levels can be measured using a variety of assays, including, for example, commercially available kits.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction.

The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., flatman et al, j.chromatogr.b848:79-87 (2007).

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-CD 47 antibody or antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or antigen-binding fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in the reference polypeptide sequence, after the sequences are aligned (and gaps introduced, if necessary) to obtain the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

When referring to percentages of sequence identity in the present application, these percentages are calculated over the full length of the longer sequence, unless otherwise specifically indicated. The full length calculation relative to the longer sequence applies to both nucleic acid and polypeptide sequences.

The terms "red blood cell" and "red blood cell" are synonymous and are used interchangeably.

The term "agglutination" refers to clumping of cells, while the term "hemagglutination" refers to clumping of a particular type of cells (i.e., red blood cells). Therefore, hemagglutination is a type of agglutination.

1.2 anti-CD 47 antibodies of the invention

Unless otherwise indicated, the terms "integrin-associated protein (IAP)," CD47, "when used herein, refer to any native CD47 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed CD47 as well as any form of CD47 or any fragment thereof resulting from intracellular processing. The term also includes variants of naturally occurring CD47, e.g., splice variants or allelic variants.

The term "anti-CD 47 antibody", "anti-CD 47", "CD47 antibody" or "antibody that binds to CD47" refers to an antibody that is capable of binding to CD47 protein or a fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting CD47. In one embodiment, the anti-CD 47 antibody binds to an unrelated, non-CD 47 protein to less than about 10% of the binding of the antibody to CD47, as measured, for example, by Radioimmunoassay (RIA). In some embodiments, the anti-CD 47 antibodies provided herein have a dissociation constant (Kd) of less than 1 μ M, less than 100nM, less than 10nM, less than 1nM, less than 0.1nM, less than 0.01nM, or less than 0.001nM (e.g., 10 nM) ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 M1。

In some embodiments, the anti-CD 47 antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions, or deletions. In preferred embodiments, the substitution, insertion or deletion occurs in a region outside the CDRs (e.g., in the FR). Optionally, the anti-CD 47 antibodies of the invention comprise post-translational modifications to the light chain variable region, the heavy chain variable region, the light chain or the heavy chain.

The CD47 antibodies provided herein exhibit inhibitory activity, e.g., inhibiting expression of CD47 (e.g., inhibiting expression of CD47 on the cell surface), activity, and/or signaling, or interfering with the interaction between CD47 and sirpa. The CD47 antibodies provided herein fully or partially reduce or modulate CD47 expression or activity upon binding to or interacting with CD47 (e.g., human CD 47). The reduction or modulation of CD47 biological function is complete, significant or partial upon interaction between the antibody and the human CD47 polypeptide and/or peptide. An antibody described herein is considered to be capable of completely inhibiting the expression or activity of CD47 when the level of expression or activity of CD47 is reduced by at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) in the presence of the antibody as compared to the level of expression or activity of CD47 in the absence of interaction (e.g., binding) with the antibody. The CD47 antibody is considered to be capable of significantly inhibiting the expression or activity of CD47 when the level of expression or activity of CD47 is reduced by at least 50% (e.g., 55%, 60%, 75%, 80%, 85%, or 90%) in the presence of the CD47 antibody as compared to the level of expression or activity of CD47 in the absence of binding to the CD47 antibody described herein. The antibody is considered to be capable of partially inhibiting the expression or activity of CD47 when the level of expression or activity of CD47 is reduced by less than 95% (e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90%) in the presence of the antibody as compared to the level of expression or activity of CD47 in the absence of interaction (e.g., binding) with the antibody described herein.

In certain embodiments, one or more amino acid modifications may be introduced in the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thio mabs," in which one or more residues of the antibody are substituted with a cysteine residue.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the invention encompasses fragments of anti-CD 47 antibodies. Examples of antibody fragments include, but are not limited to, fv, fab '-SH, F (ab') ₂ Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment to yield F (ab') ₂ A fragment that has two antigen binding sites and is still capable of cross-linking antigens.

In some embodiments, the anti-CD 47 antibodies of the invention are humanized antibodies. Different methods for humanizing antibodies are known to the skilled artisan, as reviewed by Almagro & Fransson, the contents of which are incorporated herein in their entirety by reference (Almagro JC and Fransson J (2008) Frontiers in bioscience13: 1619-1633). Almagro & Fransson distinguishes between rational and empirical approaches. Rational approaches are characterized by generating a few engineered antibody variants and evaluating their binding or any other property of interest. If the design variation does not produce the desired result, a new round of design and integration evaluation is initiated. Rational approaches include CDR grafting, resurfacing (Resurfacing), super-humanization (superanimanization), and herringbone string content Optimization (Human stripingcontent Optimization). In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high throughput screening. Thus, empirical approaches rely on reliable selection and/or screening systems that enable the search for a large number of antibody variants. In vitro display techniques, such as phage display and ribosome display, fulfill these requirements and are well known to the skilled person. Empirical approaches include FR library, guided selection (Guided selection), framework shuffling (Framework-shuffling), and Humaneering.

In some embodiments, the anti-CD 47 antibodies of the invention are human antibodies. Human antibodies can be made using various techniques known in the art. Human antibodies are generally described in van Diik and van de Winkel, curr. 368-74 (2001) and Lonberg, curr. Opin. Immunol 20:450-459 (2008).

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding characteristics. These Methods are described, for example, in Hoogenboom et al, methods in Molecular Biology 178:1-37 (compiled by O' Brien et al, human Press, totowa, NJ, 2001), and further to the methods described in, for example, mcCafferty et al, nature348:552 to 554; clackso et al, nature 352:624-628 (1991); marks et al, j.mol.biol.222:581-597 (1992); marks and Bradbury, methods in Molecular Biology248:161-175 (Lo eds., human Press, totowa, NJ, 2003); sidhu et al, j.mol.biol.338 (2): 299-310 (2004); lee et al, j.mol.biol340 (5): 1073-1093 (2004); fellouse, proc.natl.acad.sci.usa 101 (34): 12467-12472 (2004); and Lee et al, j.immunol.methods284 (1-2): 119-132 (2004).

Suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (particularly grafted with CDRs), deimmunized, or human antibodies, fab fragments, fab 'fragments, F (ab') ₂ Fragments, fragments produced by Fab expression libraries, fd, fv, disulfide linked Fv (dsFv), single chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger p. Et al (1993) proc.natl.acad.sci.u.s.a.90 (14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the foregoing.

In some embodiments, the antibodies of the invention may be monospecific, bispecific, or multispecific. Multispecific mabs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., tutt et al (1991) j. Immunol.147:60-69. The anti-CD 47 mab may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecules, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or more binding specificities.

In some embodiments, the antibodies of the invention bind to human CD47 protein.

1.3 nucleic acids of the invention and host cells comprising the same

In one aspect, the invention provides a nucleic acid encoding any of the above anti-CD 47 antibodies or fragments thereof. The nucleic acid may encode an amino acid sequence comprising a light chain variable region and/or a heavy chain variable region of an antibody, or an amino acid sequence comprising a light chain and/or a heavy chain of an antibody.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237,5,789,199 and 5,840,523, and also Charlton, methods in Molecular Biology, volume 248 (b.k.c. lo, editors, humana Press, totowa, NJ, 2003), pages 245-254, which describe expression of antibody fragments in e.coli. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. Biotech.22:1409-1414 (2004), and Li et al, nat. Biotech.24:210-215 (2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293 or 293 cells, as described, e.g., in Graham et al, J.Gen Virol.36:59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc. Natl. Acad. Sci. USA 77 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., yazaki and Wu, methods in Molecular Biology, volume 248 (b.k.c.lo, ed., humana Press, totowa, NJ), pages 255-268 (2003).

In one embodiment, a method of making an anti-CD 47 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of an anti-CD 47 antibody, nucleic acid encoding the antibody (e.g., the antibody described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody).

1.4 pharmaceutical compositions and pharmaceutical formulations

The invention also provides pharmaceutical compositions comprising one or more monoclonal antibodies that bind to CD47 or an immunologically active fragment thereof. It is to be understood that the anti-CD 47 antibodies or pharmaceutical compositions provided herein may be incorporated into suitable vehicles, excipients, and other agents in formulations for co-administration to provide improved transfer, delivery, tolerance, and the like.

The term "pharmaceutical composition" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, as well as avoiding recurrence of the associated disease.

In some embodiments, the invention also encompasses anti-CD 47 monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxic agent or an immunosuppressive agent. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see, e.g., WO05/103081. For example, cytotoxic agents include, but are not limited to: radioisotope (e.g., at) ²¹¹ ，I ¹³¹ ，I ¹²⁵ ，Y ⁹⁰ ，Re ¹⁸⁶ ，Re ¹⁸⁸ ，Sm ¹⁵³ ，Bi ²¹² ，p ³² ，pb ²¹² And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate (methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorambucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; an antibiotic; toxins such as small molecule toxins or of bacterial, fungal, plant or animal originEnzymatically active toxins, including fragments and/or variants thereof; and various known antitumor or anticancer agents.

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising anti-CD 47 antibodies and compositions comprising polynucleotides encoding anti-CD 47 antibodies. In certain embodiments, the compositions comprise one or more antibodies that bind CD47 or one or more polynucleotides encoding one or more antibodies that bind CD47. These compositions may also contain suitable pharmaceutically acceptable carriers such as pharmaceutically acceptable excipients known in the art, including buffers.

The pharmaceutical compositions of the invention may comprise an antibody of the invention and a pharmaceutically acceptable carrier. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit.

Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical press, london, chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

Pharmaceutical formulations comprising the anti-CD 47 antibodies described herein may be prepared by mixing the anti-CD 47 antibodies of the invention with the desired purity, preferably in the form of a lyophilized formulation or an aqueous solution, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Eds. (1980)).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it would be desirable to also provide statins. The active ingredients are suitably present in combination in an amount effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

1.5 methods of treatment and uses of antibodies

In one aspect, the invention relates to a method of inhibiting, antagonizing CD47 binding to sirpa in a subject, the method comprising administering to the subject an effective amount of any of the anti-CD 47 antibodies described herein, or fragment thereof. In another aspect, the invention relates to a method of promoting phagocytosis of phagocytes in a subject, comprising administering to the subject an effective amount of any of the anti-CD 47 antibodies or fragments thereof described herein. In one aspect, the invention relates to a method of treating a CD 47-targeted therapeutic related disease, comprising administering to a subject an effective amount of any of the anti-CD 47 antibodies or fragments thereof described herein. In one aspect, the invention relates to methods of ameliorating, slowing, inhibiting or preventing any disease or disorder that may be ameliorated, inhibited or prevented by eliminating, inhibiting or reducing the binding of CD47 to sirpa. In another aspect, the invention provides methods of treating a cancer or tumor in a subject, methods of alleviating a symptom of a cancer or tumor in a subject, and methods of avoiding a recurrence of a tumor or cancer in a subject by administering to a subject in need thereof an anti-CD 47 antibody or fragment thereof of the invention.

In one aspect, the anti-CD 47 antibodies and antigen-binding fragments thereof provided herein and pharmaceutical compositions comprising the same can be used as therapeutic agents for diagnosing, prognosing, monitoring, treating, alleviating and/or preventing diseases and disorders associated with aberrant CD47 expression, activity and/or signaling in a subject. The anti-CD 47 antibodies and antigen-binding fragments thereof disclosed herein and pharmaceutical compositions comprising the same can be administered by identifying the presence of diseases and disorders associated with aberrant CD47 expression, activity, and/or signaling in a subject using standard methods.

In other aspects, the invention provides the use of an anti-CD 47 antibody in the manufacture or preparation of a medicament for the treatment of a related disease or disorder as mentioned above.

In certain embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent, such as a chemotherapeutic agent, a radiotherapeutic agent, or a biomacromolecule drug. In one embodiment, the biomacromolecule drug is, for example, a variety of monoclonal antibody drugs that attack tumor cells through T cell recognition, such as rituximab, cetuximab, and trastuzumab.

Such combination therapies include combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, wherein administration of the anti-CD 47 antibodies of the invention can occur before, simultaneously with, and/or after administration of additional therapeutic agents and/or adjuvants.

The antibodies of the invention (and any additional therapeutic agent) may be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if required for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

In another aspect, the antibodies of the invention can be used to detect the progress of a treatment for a CD 47-associated disease in vivo or in vitro, e.g., by measuring an increase or decrease in the number of CD 47-expressing cells (e.g., cancer cells), it can be determined whether a particular therapy aimed at treating the disease, alleviating the symptoms, is effective.

Most CD47 antibodies are reported to cause hemagglutination of human erythrocytes. Hemagglutination is an example of a homotypic interaction, where treatment with a bivalent CD 47-binding entity causes two CD 47-expressing cells to aggregate or agglutinate. For example, as a whole IgG or F (ab') ₂ The CD47 antibody MABL is reported to cause hemagglutination of erythrocytes, and this effect is attenuated only when MABL becomes scFv or bivalent scFv (see, e.g., unos, kinoshita Y, azuma Y, etc., antibody activity of monoclonal antibody against CD47 in xenogram models of human leukamia, oncol Rep 2007 17 1189-94, kikuchi Y, unos, yoshimura Y, etc., abivalent single-chain Fv fragment against CD47 antigens for leucocytic cells, biochem Res 2004 912-8. Other known CD47 antibodies (including B6H12, BRC126 and CC2C 6) also cause hemagglutination of RBCs. Therefore, agglutination of cells is a major limitation for the therapeutic targeting of CD47 using existing whole IgG antibodies.

In view of the fact that most of the antibodies disclosed in the prior art blocking the interaction of CD47 with sipra to promote phagocytosis cause significant cell agglutination problems, there is still an urgent need to obtain new anti-CD 47 antibodies that are not only capable of effectively promoting phagocytosis by macrophages but also do not cause cell agglutination. The antibody disclosed in the present application satisfies the need in that it is not only effective in promoting phagocytosis, but even has excellent anti-tumor growth and tumor-eliminating effects, but also at the same time the anti-CD 47 antibody disclosed in the present application does not significantly induce cell agglutination while being treated, and thus has significantly reduced side effects.

The level of agglutination can be quantified by routine experimentation by one skilled in the art, for example hemagglutination of RBCs. For example, one skilled in the art can perform a hemagglutination assay in the presence of a CD47 antibody of the present invention, followed by measuring the area of the RBC dot to determine the level of hemagglutination, as described in the examples below. In some cases, the RBC spot area in the presence of a CD47 antibody of the invention was compared to the RBC spot area in the absence of a CD47 antibody of the invention (i.e., under zero hemagglutination conditions), as well as the RBC spot area in the presence of other known CD47 antibodies. In this manner, hemagglutination was quantified relative to a baseline control. A larger area of RBC spot indicates a higher level of hemagglutination. Alternatively, hemagglutination can also be quantified using densitometric analysis of RBC spots.

1.6 methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-CD 47 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CD47 in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue.

In certain embodiments, labeled anti-CD 47 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary indicia include, but are not limited toRadioactive isotope ³² P、 ¹⁴ C、 ¹²⁵ I、 ³ H and ¹³¹ i, fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β -galactosidase, glucoamylase, lytic enzymes, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes utilizing a hydrogen peroxide oxidation dye precursor such as HR, lactoperoxidase, or microperoxidase (microperoxidase), biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

The following examples further illustrate the invention, however, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

1.7 sequences of exemplary anti-CD 47 antibodies of the invention

Full-length amino acid sequences of heavy chain and light chain of antibody

ADI26624-IgG4

HC amino acid sequence (SEQ ID NO: 74)

LC amino acid sequence (SEQ ID NO: 75)

ADI29336-IgG4

HC amino acid sequence (SEQ ID NO: 76)

LC amino acid sequence (SEQ ID NO: 75)

ADI29340-IgG4

HC amino acid sequence (SEQ ID NO: 77)

LC amino acid sequence (SEQ ID NO: 75)

ADI26630-IgG4

HC amino acid sequence (SEQ ID NO: 78)

LC amino acid sequence (SEQ ID NO: 79)

ADI29341-IgG4

HC amino acid sequence (SEQ ID NO: 80)

LC amino acid sequence (SEQ ID NO: 79)

ADI 29349-IgG4

HC amino acid sequence (SEQ ID NO: 81)

LC amino acid sequence (SEQ ID NO: 79)

ADI 26591-IgG4

HC amino acid sequence (SEQ ID NO: 82)

LC amino acid sequence (SEQ ID NO: 83)

ADI 29371-IgG4

HC amino acid sequence (SEQ ID NO: 84)

LC amino acid sequence (SEQ ID NO: 83)

ADI 30793-IgG4

HC amino acid sequence (SEQ ID NO: 85)

LC amino acid sequence (SEQ ID NO: 86)

ADI 30794-IgG4

HC amino acid sequence (SEQ ID NO: 87)

LC amino acid sequence (SEQ ID NO: 86)

ADI26624-IgG1

HC amino acid sequence (SEQ ID NO: 88)

LC amino acid sequence (SEQ ID NO: 75)

ADI29336-IgG1

HC amino acid sequence (SEQ ID NO: 89)

LC amino acid sequence (SEQ ID NO: 75)

ADI29340-IgG1

HC amino acid sequence (SEQ ID NO: 90)

LC amino acid sequence (SEQ ID NO: 75)

ADI26630-IgG1

HC amino acid sequence (SEQ ID NO: 91)

LC amino acid sequence (SEQ ID NO: 79)

ADI29341-IgG1

HC amino acid sequence (SEQ ID NO: 92)

LC amino acid sequence (SEQ ID NO: 79)

ADI 29349-IgG1

HC amino acid sequence (SEQ ID NO: 93)

LC amino acid sequence (SEQ ID NO: 79)

ADI 26591-IgG1

HC amino acid sequence (SEQ ID NO: 94)

LC amino acid sequence (SEQ ID NO: 83)

ADI 29371-IgG1

HC amino acid sequence (SEQ ID NO: 95)

LC amino acid sequence (SEQ ID NO: 83)

ADI 30793-IgG1

HC amino acid sequence (SEQ ID NO: 96)

LC amino acid sequence (SEQ ID NO: 86)

ADI 30794-IgG1

HC amino acid sequence (SEQ ID NO: 97)

LC amino acid sequence (SEQ ID NO: 86)

Amino acid sequence of CD47 protein (SEQ ID NO: 56)

The sequences for the negative controls appearing in the figures are as follows:

IgG1 HC：

IgG1 LC

IgG4 HC：

IgG4 LC：

when the experimental antibody is of the IgG1 type, the negative control is IgG 1; when IgG4 type was used as the experimental antibody, igG4 was used as the negative control.

Examples

Example 1 production and purification of anti-CD 47 antibodies and control antibodies

The amino acid sequences of the CDR regions, the light and heavy chain variable regions, the light and heavy chains of the 10 antibodies exemplified by the present invention (ADI-26624, ADI-29336, ADI-29340, ADI-26630, ADI-29341, ADI-29349, ADI-26591, ADI-29371, ADI-30793, ADI-30794), as well as the corresponding nucleotide sequences are listed in the "sequence Listing" section of the present application. In addition, the above-described exemplary antibodies of the present invention have the light chain, heavy chain, light chain variable region and heavy chain variable region with the sequence numbers shown in Table 1.

The antibodies of the invention were expressed and purified in yeast as well as CHO-S cells.

Expression and purification in yeast:

yeast-based antibody presentation libraries (Adimab) were amplified according to the existing methods (WO 2009036379; WO 2010105256 W02012009568), with a diversity of 1X 10 per library ⁹ . Briefly, the first two rounds of screening were performed using the MACS system from Miltenyi for magnetically activated cell sorting. First, yeast cells (. About.1X 10) of the library were cultured ¹⁰ Cells/library) were incubated in FACS wash buffer (phosphate buffer containing 0.1% bovine serum albumin) containing 100nM biotin-labeled CD47 antigen (Acro Biosystems, cat. No. CD7-H5227-1 mg) for 15 minutes at room temperature, respectively. One wash with 50ml of pre-cooled FACS wash buffer, resuspend the cells with 40ml of the same wash buffer and incubate at 4 ℃ for 15 min with 500. Mu.l streptomycin microbeads (Miltenyi LS). After centrifugation at 1000rpm for 5min and discarding the supernatant, the cells were resuspended with 5ml FACS wash buffer and the cell solution was applied to a Miltenyi LS column. After the loading was complete, the column was washed 3 times with 3ml each time of FACS wash buffer. The Miltenyi LS column was removed from the magnetic field and grown in 5mlEluted yeast cells were collected and grown overnight at 37 ℃.

The next round of sorting was performed using a flow cytometer: approximately 1X 10 obtained by screening through MACS system ⁸ The yeast cells of (2) were washed three times with FACS buffer and cultured at room temperature in CD47 antigen containing a low concentration of biotin (100-1 nM) marker. After discarding the culture solution and washing the cells twice with FACS wash buffer, the cells were mixed with LC-FITC (FITC-labeled goat anti-human immunoglobulin F (ab') kappa chain antibody, southern Biotech) (1: 100 dilution) and SA-633 (streptavidin-633, molecular Probes) (1: 500 dilution) or SA-PE (streptavidin-phycoerythrin, sigma) (1: 50 dilution) reagents and incubated at 4 ℃ for 15 minutes. The cells were eluted twice with pre-cooled FACS wash buffer and resuspended in 0.4ml buffer and transferred to a separation tube with filter. Cells were sorted using FACS ARIA (BD Biosciences).

The yeast cells expressing the anti-CD 47 antibody obtained by the screening were induced by shaking at 30 ℃ for 48 hours to express the anti-CD 47 antibody. After the induction was completed, the yeast cells were removed by centrifugation at 1300rpm for 10min, and the supernatant was harvested. The anti-CD 47 antibody in the supernatant was purified by Protein A, eluted with acetic acid solution of pH2.0, and the anti-CD 47 antibody was harvested with an antibody purity of > 95%. The corresponding Fab fragments were obtained by digestion with papain and purification with KappaSelect (GE Life medical group).

Expression and purification in CHO-S cells:

according to manufacturer's instructions, useThe kit (Invitrogen) generated a CHO-S cell line expressing the antibody. The DNA sequences of the heavy and light chains of the antibody molecule were first inserted into the same pcho1.0 plasmid, with the heavy chain upstream of the light chain. The constructed pCHO1.0 plasmid was then transferred into a CHO cell line by chemical transfection and electrotransfection, and antibody production was measured by ForteBio to determine transfection efficiency 48 hours after transfection. After transfection, two rounds of pressure screening are carried out on the cells to obtain a cell pool (pool) with high expression of antibody. Then expanding the cell pool to a large numberAntibody expression and cell supernatants were collected and the supernatants were purified with Protein A to achieve antibody purity > 95%.

TABLE 1 amino acid sequence numbering of the light chain, heavy chain, light chain variable region and heavy chain variable region of 10 exemplary antibodies obtained in the present invention.

The following control antibodies used in the examples were expressed and purified in 293HEK cells:

control antibody
	Hu5F9
AB6.12

Hu5F9 is a human CD47 antibody transiently expressed in 293HEK cells, and has a sequence identical to that of antibody "5F9" in US patent US2015/0183874 A1. AB6.12 is a humanized CD47 antibody transiently expressed in 293HEK cells, the sequence of which is identical to that of antibody "AB6.12" in US 9045541. The antibody "AB6.12" disclosed in US 9045541 is a CD47 antibody that will not cause significant agglutination of cells.

For transient expression of the antibody in 293HEK cells, the vector pTT5 was used. The heavy and light chains of the antibody were first cloned into separate pTT5 vectors. The pTT5 vector carrying the heavy and light chains of the antibody molecule was transferred into 293HEK cells using chemical transfection. Chemical transfection assay usedThe agent was PEI (purchased from Polysciences) transiently transfected with cultured 293HEK according to the protocol provided by the manufacturer. Firstly, plasmid DNA and a transfection reagent are prepared in an ultra-clean bench, half of an F17 culture medium (Gibco) (the volume is 1/5 of the transfection volume) is added into a 50ml centrifuge tube, one part of the culture medium is added with filtered plasmids (130 mu g/100 ml), the other part of the culture medium is added with filtered PEI (1 g/L, polysciences) (the mass ratio (plasmids: PEI) = 1: 3), the mixture is mixed for 5min, and the two are mixed softly and evenly for 20 times and are kept stand for 15-30min, which is not more than 30min. Gently pour DNA/PEI mix into 293HEK cells and mix well, 8% CO at 37 ℃% ₂ The cells were cultured for 7 days under the condition of (1), and fresh medium was fed every 48 hours. After 7 days or when the cell viability is less than or equal to 60 percent after continuous culture, centrifuging for 20min at 13000 rpm. The supernatant was taken and purified with Protein A to give antibody purity > 95%.

Example 2: affinity assay for anti-CD 47 antibodies of the invention

The equilibrium dissociation constant (KD) for binding of the above 10 exemplary antibodies of the invention (Fab fragment was used in monovalent experiments to exclude the possible effect of Fc fragment) to human CD47 (hCD 47) was determined using a biophotonic interferometry (ForteBio) assay.

ForteBio affinity assays were performed according to the current protocol (Estep, P et al, high throughput solution Based measurement of antibody-antibody affinity and epitope binding. MAbs,2013.5 (2): p.270-8). Briefly, the sensor was equilibrated in assay buffer for 30 minutes under the line, then the baseline was established by on-line detection for 60 seconds, and ForteBio affinity measurements were performed on an AHQ sensor (ForteBio) on-line loading of purified antibody obtained as described above. The sensor with the loaded antibody was exposed to 100nM of CD47 antigen for an additional 5 minutes before transferring the sensor to assay buffer for dissociation for 5 minutes for dissociation rate measurements. Kinetic analysis was performed using a 1: 1 binding model.

In experiments performed as described in the above assays, ADI-26624, ADI-26630, ADI-26591, ADI-29336, ADI-29340, ADI-29341, ADI-29349, ADI-29371, ADI-30793, and ADI-30794 affinities are shown in Table 2.

Table 2: measurement of binding of an antibody of the invention in IgG1 form by Bio-optical interferometry

Note: n.b. indicates no binding.

It can be seen that all of the above 10 exemplary antibodies of the present invention showed extremely high affinity, which is comparable to the excellent CD47 antibody Hu5F9 known and recognized in the art.

Example 3: binding of the anti-CD 47 antibodies of the invention to human CD47

The binding of the above 10 exemplary antibodies of the invention to human CD47 was measured in a flow cytometry-based assay.

CHO cells overexpressing human CD47 (CHO-hCD 47 cells) were generated by transfection of pCHO1.0 vector (Invitrogen) carrying human CD47cDNA (Sino Biological) cloned into the Multiple Cloning Site (MCS). CHO-hCD47 cells (0.2X 10) ⁶ Individual cells) were incubated with varying concentrations of the experimental antibody ((10 exemplary antibodies of the invention described above and Hu5F 9) at a maximum concentration of 900nM, three-fold dilution, total 11 concentrations tested) in PBS containing 0.1% Bovine Serum Albumin (BSA) for 30 minutes on ice. The cells were then washed at least twice and incubated with a secondary antibody (PE-labeled goat anti-human IgG antibody, southern Biotech, final concentration 5. Mu.g/ml) in PBS containing 0.1% BSA for 30 minutes on ice (protected from light). Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and concentration dependent curves were fitted with GraphPad according to its MFI.

ADI-26591, ADI-26624 and ADI-26630 (IgG 1 format, expressed in yeast) bound hCD47 (SEQ ID NO: 56) overexpressed on CHO cells with EC50 values of 3.77nM, 2.254nM and 3.895nM, respectively, consistent with the binding capacity of the control antibody Hu5F9 to hCD47 overexpressed on CHO cells (the EC50 value of the control antibody Hu5F9 was 3.726 nM) (see FIG. 1).

In experiments performed as described in the test methods above, the IgG1 forms of ADI-29336, ADI-29340, ADI-29341, ADI-29349, ADI-29371, ADI-30793, and ADI-30794 produced in yeast bound hCD47 overexpressed on CHO cells with EC50 values of 6.725nM, 3.529nM, 3.344nM, 3.13nM, 2.132nM, 2.921nM, and 3.697nM, respectively, essentially corresponding to the binding capacity of the control antibody Hu5F9 to CD47 overexpressed on CHO cells (EC 50 value of 3.726 nM).

Antibodies in the form of IgG4 produced in CHO cells ADI-29336, ADI-29340, ADI-29341, ADI-29349 and ADI-29371 bound hCD47 overexpressed on CHO cells with EC50 values of 2.475nM, 2.194nM, 1.892nM, 2.043nM and 2.31nM, respectively, all having a higher affinity for hCD47 at the cellular level than the control antibody Hu5F9 (EC 50 value of 3.726 nM).

Example 4 blocking of the interaction of human CD47 ligand SIRP alpha with CD47 by the anti-CD 47 antibodies of the invention

The ability of the above 10 exemplary antibodies of the invention to block human CD47 binding to sirpa was measured by flow cytometry.

0.2X 10 prepared as described in example 3 above ⁶ Human CD47 expressing CHO cells were incubated with experimental antibodies ((ADI-26624, ADI-26630, ADI-29336, ADI-29340, ADI-29341, ADI-29349, and Hu5F 9), maximum concentration 900nM, three-fold dilution, total 11 concentrations tested) and 200nM SIRPa protein labeling mouse Fc (Acrobiosystems) in PBS containing 0.1% BSA on ice for 30min. The cells were then washed 3 times and subsequently incubated for 30 minutes on ice (protected from light) in PBS containing 0.1% BSA using a goat anti-mouse IgG-APC (allophycocyanin) secondary antibody (Biolegend). Cells were washed 3 times. Flow cytometry detection was performed on the Accuri C6 system (BD Biosciences) and MFI was calculated on the C6 software.

The ability of ADI-26624, ADI-29336, ADI-29340, ADI-29371, ADI-26630, ADI-29341, and ADI-29349 as IgG1 produced in yeast cells to block the binding of human SIRP α -APC to CD47 is consistent with the ability of the control antibody AB 6.12.

Specifically, the IC50 of ADI-26624, ADI-29336, and ADI-29340 ability to block the binding of human SIRPa-APC to CD47 were 11.2nM, 8.548nM, and 5.081nM, respectively. ADI-26630, ADI-29341, ADI-29349 blocked the ability of human SIRP α -APC to bind CD47 with an IC50 of 2.986nM, 2.476nM, 3.097nM, respectively. The IC50 of the control antibody AB6.12 for its ability to block the binding of human sirpa-APC to CD47 is 3.385nM. (see FIG. 2).

Antibodies ADI-26624, ADI-26630, ADI-29336, ADI-29340, ADI-29341, and ADI-29349 in the IgG4 format produced in CHO cells all blocked the binding of human SIRP α -APC to CD47 slightly more than the control antibodies AB6.12 and Hu5F9.

Specifically, the IC50 of ADI-26624, ADI-29336 and ADI-29340 for blocking the ability of human SIRP alpha-APC to bind CD47 are 1.043nM, 1.389nM and 1.223nM, respectively. ADI-26630, ADI-29341, ADI-29349 blocked the ability of human SIRP α -APC to bind CD47 with IC50 of 1.123nM, 0.6042nM, 0.7355nM, respectively. The IC50 for the control antibodies AB6.12 and Hu5F9, the ability to block the binding of human sirpa-APC to CD47, is 1.768nM and 1.843nM, respectively. (see FIG. 3).

Example 5 detection of the ability of the anti-CD 47 antibody of the present invention to promote phagocytosis of tumor cells by macrophages

The ability of the antibodies of the invention (ADI-26624, ADI-29336, ADI-29340, ADI-26630, ADI-29341, ADI-29349, ADI-29371, ADI-30793 and ADI-30794) to promote phagocytosis of tumor cells by macrophages was measured in a flow cytometry-based assay.

Fresh blood from donors was subjected to density gradient centrifugation to obtain Peripheral Blood Mononuclear Cells (PBMCs). From the isolated PBMC according to the kit (EasySep) ^TM Human CD14 Positive Selection Kit, steam cell) Specification purified to give CD14 Positive monocytes, 10ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF, R)&D Systems) adherent culture for 7 consecutive days; on day 5, the cells were stimulated for 1 hour with 20ng/mL interferon-gamma (IFN-. Gamma., acrobiosystem) and then for 48 hours with 100ng/mL lipopolysaccharide (LPS, sigma) to induce monocytes to macrophages. Target tumor cells CCRF-CEM (purchased from ATCC) were fluorescently labeled according to the instructions of CellTrace (TM) CFSE kit. The labeled tumor cells were co-cultured with the above-mentioned differentiated macrophages at a ratio of 4: 1, and simultaneously, experimental antibodies at different concentrations were added and incubated at 37 ℃ for 3 hours. The cells were then washed at least twice, and Allophycocyanin (APC) -labeled CD14 antibody (purchased from BD) was added and incubated on ice (protected from light) in PBS containing 0.1% BSAIncubate for 30min. Cells were washed at least twice and analyzed by flow cytometry. The phagocytosed cell population is a cell population that is positive for CD14 in living cells and also positive for the fluorescent dye CFSE (carboxyfluorescein diacetate, succinimidyl ester).

ADI-29336, ADI-29340, ADI-29341, ADI-29349, ADI-30793 and ADI-30794 in IgG1 form produced in yeast cells all have strong ability to promote macrophages to phagocytose tumor cells, wherein the ability of ADI-29340 to promote macrophages to phagocytose tumor cells is stronger than that of control antibodies Hu5F9 and AB 6.12; ADI-30793 and ADI-30794 promoted phagocytosis of tumor cells by macrophages comparable to that of the control antibody AB6.12 (see FIG. 4 and FIG. 5).

ADI-26624 and ADI-26630 in the form of IgG4 produced in CHO cells were able to efficiently promote phagocytosis of tumor cells by macrophages, the ability of ADI-26624 and ADI-26630 to promote phagocytosis of tumor cells by macrophages being in agreement with the ability of the control antibody Hu5F9 (see FIG. 6).

ADI-29336, ADI-29340, ADI-29341, ADI-29349 and ADI-29371 in the form of IgG4 produced in CHO cells all have a strong ability to promote phagocytosis of tumor cells by macrophages. From the results, it can be seen that ADI-29336, ADI-29340, ADI-29341, and ADI-29349 promote the ability of macrophages to phagocytose tumor cells significantly higher than that of the control antibody Hu5F 9; ADI-29371 has a capacity to promote phagocytosis of tumor cells by macrophages comparable to that of the control antibody Hu5F9 (see FIG. 7).

Example 6 antitumor Activity of anti-CD 47 antibodies of the invention

The anti-tumor efficacy of the CD47 antibodies of the invention (ADI-26624, ADI-26630, ADI-29340 and ADI 29341) was studied in a NOD/SCID mouse model.

The method comprises the following steps:

human burkitt's lymphoma Raii cells (ATCC # CCL-86) were purchased from ATCC and routinely subcultured for subsequent in vivo experiments strictly as per ATCC requirements. Cells were harvested by centrifugation, resuspended in sterile PBS and adjusted to a cell density of 10 ⁷ Each/ml. Taking 0.1m1 cell suspension and MatrigelThe mixture was inoculated 1: 1 subcutaneously into the right side of NOD/SCID mice (Experimental animals technology, inc., wintolinhua, beijing). Tumors and body weights were measured twice weekly throughout the study, and mice were euthanized when tumors reached the endpoint or when they had > 20% weight loss. 10 days after inoculation, mice meeting experimental requirements were randomly grouped into 8 mice each. The tumor volume of the mice is measured by a vernier caliper, and the calculation formula is as follows: 2 × Length/2 of each group, maximum tumor volume of 110mm in mice of each group ³ 。

First, the applicant studied the tumor-inhibiting effect of the ADI-26624 and ADI-26630 antibodies of the present invention.

The mice obtained in the above method were randomly grouped and given different treatments: i.e., intraperitoneal injections of 1mg/kg or 5mg/kg of PBS, igG control antibody (IgG 4), benchmark (Hu 5F 9), antibodies of the invention ADI-26630 and ADI-26624 were administered once every two days for 2 weeks. The specific grouping and mode of application are as follows in table 3:

table 3:

at the end of the experiment, the tumor growth inhibition rate was calculated using the following formula:

TGI％＝100％×((Tvol _{after PBS treatment} -Tvol _{After antibody treatment} )/(Tvol _{After PBS treatment} -Tvol _{Before PBS treatment} ))，

Wherein Tvol _{After PBS treatment} Is the tumor volume, tvol, after the experiment of the blank control PBS group is finished _{After antibody treatment} Is the tumor volume, tvol, after completion of the antibody group (IgG, hu5F9 and the antibody of the present invention) experiment _{Before PBS treatment} Is the initial tumor volume of the blank control PBS group.

The results of the experiments are shown in FIG. 8, FIG. 9 and Table 4 below, and it can be seen that the anti-CD 47 monoclonal antibodies ADI-26624 and ADI-26630 expressed in the form of IgG4 in CHO cells of the present application significantly inhibited tumor growth compared to IgG control (equitech-Bio) and the control antibody Hu5F9.

The tumor growth inhibition rates of the ADI-26630 mg/kg group, the ADI-26624 1mg/kg group and the ADI-26624 5mg/kg group are respectively 100%, 104%, 79% and 94%. Of the 8 mice in the ADI-26630 mg/kg group, 5 tumors completely disappeared; of 8 mice in the ADI-26630 mg/kg group, 2 tumors completely disappeared in a number higher than that of the control antibody Hu5F9 (5 mg/kg group, 2 mice in the 1mg/kg group, 1 mouse, respectively) at the same dose (Table 4). There was no significant change in body weight 32 days after inoculation in all groups of mice in this study.

Therefore, the antibody has good tumor treatment effect, and the effect is better than that of the control group Hu5F9 antibody.

Table 4: form IgG4 expressed in CHO cells statistical table of mouse tumor size and growth inhibition rate in the anti-tumor activity study of the antibody of the present invention.

Next, the inventors continued to examine the tumor-inhibiting effect of the antibodies ADI-29340 and ADI 29341.

The mice obtained in the above-described method were randomly grouped and given different treatments, i.e., intraperitoneal injections of 0.5mg/kg or 5mg/kg of PBS, igG control antibody (IgG 4), inventive antibody ADI-26630, ADI-29340, and ADI29341, once every two days for 2 weeks. The specific grouping and mode of administration are as follows in table 5:

TABLE 5

At the end of the experiment, the tumor growth inhibition rate was calculated using the above formula, and it was found that anti-CD 47 monoclonal antibodies ADI-26630, ADI-29340, and ADI-29341, expressed in CHO cells in the form of IgG4, significantly inhibited the growth of tumors (table 6, fig. 10, and fig. 11).

The tumor growth inhibition rates of ADI-26630 0.5mg/kg, ADI-26630 mg/kg, ADI-29340.0.5 mg/kg, ADI-29340 5mg/kg, ADI-29341.0.5 mg/kg and ADI-29341 5mg/kg groups are respectively 99%, 110%, 103%, 109%, 104% and 109%. Of the 8 mice in the ADI-29340.5mg/kg group, 5 tumors completely disappeared; of 8 mice in the ADI-29341 0.5mg/kg group, 4 tumors completely disappeared; tumors of 8 mice in both ADI-26630 and ADI-29340 mg/kg groups disappeared completely (Table 6). There was no significant change in body weight in all groups of mice in this study 30 days after inoculation.

Therefore, the antibody of the invention has good tumor treatment effect.

Table 6: form IgG4 expressed in CHO cells statistical table of mouse tumor size and growth inhibition rate in the anti-tumor activity study of the antibody of the present invention.

Example 7 detection of erythrocyte agglutination-promoting Activity by anti-CD 47 antibody of the present invention

It is known in the art that most anti-CD 47 antibodies have the side effect of promoting erythrocyte agglutination, and thus their therapeutic applications are limited. To this end, the inventors further investigated the hemagglutination of the antibodies disclosed in the present application.

The detection method comprises the following steps:

fresh human blood was collected, washed three times with PBS to prepare a 10% human red blood cell suspension, and the human red blood cells were incubated with experimental antibodies (maximum concentration of 60ug/ml, three-fold dilution, total 11 dots) at 37 ℃ for 2-6 hours, and the reaction was performed in a 96-well round bottom plate. And after the reaction is finished, photographing and judging the result. The result was judged as a criterion that agglutination of erythrocytes occurred if erythrocytes sink to the bottom of the well and are tiled in a net shape (see the results of Hu5F9 in fig. 12), and erythrocytes sink to the bottom of the well in a dot shape if erythrocytes do not agglutinate (see the control in fig. 12).

In experiments performed as described in the above assay, the hemagglutination results are shown in fig. 12. As can be seen from fig. 12, the agglutination activities of the erythrocytes of ADI26630, ADI29340 and ADI29341 were weak, and the agglutination-promoting activity thereof was significantly lower than that of Hu5F9, which is equivalent to AB6.12 in the control group. It can be seen that the antibodies disclosed herein have significantly reduced hemagglutination and thus significantly reduced side effects in clinical treatment and can be widely used in the treatment of various cancers.

Claims (24)

1. An isolated anti-CD 47 monoclonal antibody or antigen-binding fragment thereof comprising 3 complementarity determining regions HCDR1, HCDR2 and HCDR3 of a heavy chain variable region, and 3 complementarity determining regions LCDR1, LCDR2 and LCDR3 of a light chain variable region, wherein:

(I) The amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 19, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 25, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 28;

(II) the amino acid sequence of HCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 9, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 19, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 25, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 28; or

(III) the amino acid sequence of HCDR1 is shown as SEQ ID NO. 5, the amino acid sequence of HCDR2 is shown as SEQ ID NO. 12, the amino acid sequence of HCDR3 is shown as SEQ ID NO. 19, the amino acid sequence of LCDR1 is shown as SEQ ID NO. 23, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 25, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 28.

2. The isolated monoclonal antibody or antigen binding fragment thereof of claim 1, comprising

(i) A heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:47 and having HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:47, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:55 and having LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:55, or

(ii) A heavy chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:48 and having HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO:48, and a light chain variable region comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:55 and having LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NO:55, or

(iii) A heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:49 having at least 90% sequence identity and having the amino acid sequences of HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:49, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:55 having at least 90% sequence identity and having the amino acid sequences of LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 55.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

(i) A heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 78 or 91 and having HCDR1, HCDR2 and HCDR3 depicted in SEQ ID NO 78, and a light chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 79 and having LCDR1, LCDR2 and LCDR3 depicted in SEQ ID NO 79, or

(ii) A heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 80 or 92 and having HCDR1, HCDR2 and HCDR3 depicted in SEQ ID NO 80, and a light chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 79 and having LCDR1, LCDR2 and LCDR3 depicted in SEQ ID NO 79, or

(iii) A heavy chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 81 or 93 and having HCDR1, HCDR2 and HCDR3 depicted in SEQ ID NO 81, and/or a light chain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence depicted in SEQ ID NO 79 and having LCDR1, LCDR2 and LCDR3 depicted in SEQ ID NO 79.

4. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a humanized or human antibody.

5. The isolated monoclonal antibody or antigen binding fragment thereof of claim 1 or 2, wherein the antigen binding fragment is selected from the group consisting of Fab, fab '-SH, fv, scFv, or (Fab') ₂ And (3) fragment.

6. The isolated monoclonal antibody, or antigen-binding fragment thereof, of claim 1 or 2, comprising a framework sequence, wherein at least a portion of the framework sequence is a human consensus framework sequence.

7. An isolated nucleic acid encoding the isolated anti-CD 47 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6.

8. A vector comprising the nucleic acid of claim 7.

9. The vector of claim 8, which is an expression vector.

10. A host cell comprising the vector of claim 8 or 9.

11. The host cell of claim 10, which is a prokaryotic cell or a eukaryotic cell.

12. The host cell of claim 10, which is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for the production of an antibody or antigen-binding fragment thereof.

13. The host cell of claim 12, which is a CHO cell or 293 cell.

14. A method of making an anti-CD 47 monoclonal antibody, or antigen-binding fragment thereof, comprising culturing the host cell of claim 10 under conditions suitable for expression of a nucleic acid encoding the anti-CD 47 monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 1 to 6.

15. The method of claim 14, further comprising the step of recovering said anti-CD 47 monoclonal antibody or antigen-binding fragment thereof from said host cell.

16. An anti-CD 47 monoclonal antibody or antigen-binding fragment thereof prepared by the method of claim 14 or 15.

17. A pharmaceutical composition comprising the anti-CD 47 antibody, or antigen-binding fragment thereof, of any one of claims 1 to 6 and 16, and optionally a pharmaceutically acceptable carrier.

18. Use of the anti-CD 47 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and 16 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for the treatment of cancer or tumor.

19. Use of the anti-CD 47 monoclonal antibody or antigen-binding fragment thereof of any one of claims 1 to 6 and 16 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for alleviating a symptom of a cancer or tumor.

20. The use of claim 18 or 19, wherein the cancer or tumor is various hematological and solid tumors.

21. The use of claim 20, wherein the cancer or tumor is acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-hodgkin's lymphoma, multiple myeloma, lymphoma, breast cancer, head and neck cancer, gastric cancer, lung cancer, intestinal cancer, ovarian cancer, liver cancer, renal cancer, pancreatic cancer, bladder cancer, colorectal cancer, glioma, melanoma.

22. The use of any one of claims 18 to 19, wherein the medicament may be combined with one or more other medicaments.

23. The use of claim 22, wherein the other drugs are rituximab, cetuximab, and trastuzumab.

24. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 and 16 in the preparation of a test reagent for detecting the presence of CD47 protein in a sample.